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United States Patent |
6,096,323
|
Walker
,   et al.
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August 1, 2000
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Vaccine against papillomatous digital dermatitis (PDD)
Abstract
This invention relates to the diagnosis and prevention of ungulate diseases
caused by the spirochete bacteria Treponema. The invention specifically
relates to isolated cultures of this spirochete and isolated nucleic acids
and proteins.
Inventors:
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Walker; Richard L. (Davis, CA);
Read; Deryck H. (Yucaipa, CA);
Hird; David W. (Davis, CA);
Lefebvre; Rance B. (Davis, CA);
Berry; Steven L. (Davis, CA);
Cullor; James S. (Woodland, CA);
Lefler; Hank M. (Reno, NV)
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Assignee:
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The Regents of the University of California (Oakland, CA)
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Appl. No.:
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191099 |
Filed:
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November 12, 1998 |
Current U.S. Class: |
424/262.1; 424/93.1; 424/93.4; 424/184.1; 424/234.1; 424/823; 435/243; 435/252.1 |
Intern'l Class: |
A01N 063/00; A61K 039/00; A61K 039/02; C12N 001/00 |
Field of Search: |
424/184.1,234.1,262.1,93.1,93.4,823
435/243,252.1
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References Cited
Other References
Bassett et al. Vet. Rec. 126: 164-165, 1990.
Walker et al. Vet Micro. 47: 343-355, 1995.
Walker et al. AJVR 58(7): 744-748, 1997 (Jul.).
Choi et al. Int'l J. of Syst. Bact 47(1):175-181, 1997 (Jan.).
Hygieia Biological Laboratories, (1996) "Papillomatous Digital Dermatitis
Treatment using Serpens ssp. bacterin" (Product information).
Addie, D.D., et al. (1990) "Control of feline coronavirus infection in
kittens", Veterinary Record 126:164-165.
Blowey, R.W., et al. (1988) "Digital dermatitis in dairy cattle",
Veterinary Record 122:505-508.
Blowey, R.W., et al. (1994) "Observations on the pathogenesis of digital
dermatitis in cattle" Veterinary Record, 135:115-117.
Choi, B.-K., et al. (1997) "Spirochetes from Digital Dermatitis Lesions in
Cattle Are Closely Related to Treponemes Associated with Human
Periodontitis", International Journal of Systematic Bacteriology
47(1):157-181.
Dopfer, D., et al. (1997) "Histological and bacteriological evaluation of
digital dermatitis in cattle, with special reference to spirochaetes and
Campylobacter faecalis", Veterinary Record, 140:620-623.
Read, D.H., et al. (1992) "An invasive spirochaete associated with
interdigital papillomatosis of dairy cattle", Veterinary Record 130:59-60.
Read, D.H., et al. (1995) "Studies on the Etiology of Papillomatous Digital
Dermatitis (Footwarts) of Dairy Cattle", 38th Annual Meeting--American
Association of Veterinary Laboratory Diagnosticians (Abstracts) 68.
Read, D.H., et al. (1996) "Experimental transmission of Papillomatous
Digital Dermatitis (Footwarts) in Cattle" (Abstract), Vet. Pathol
33:(5):607 (No. 151).
Rebhun, William C., et al. (1980) "Interdigital Papillomatosis in Dairy
Cattle", JAVMA 177(5):437-440.
Rijpkema, S.G.T., et al. (1997) "Partial identification of spirochaetes
from two dairy cows with digital dermatitis by polymerase chain reaction
analysis of the 16S robosomal RNA gene", Veterinary Record 140:257-259.
Scavia, G., et al. (1994) "Digital Dermatitis: Further contributions on
clinical and pathological aspects in some herds in northern Italy", Eighth
International Symposium on Disorders of the Ruminant Digit and
International Conference on Bovine Lameness (Proceedings and Abstracts),
174-175.
Zemljic, Borut (1994) "Current Investigations into the Cause of Dermatitis
Digitalis in Cattle", Eighth International Symposium on Disorders of the
Ruminant Digit and International Conference on Bovine Lameness
(Proceedings and Abstracts), 164-167.
Walker, R.L., et al. (1996) "Humoral Response of Dairy Cattle to
Spirochetes Associated with Papillomatous Digital Dermatitis", The
Conference of Research Workers in Animal Diseases--Abstracts, No. 36.
Walker, R.L., et al. (1995) "Spirochetes isolated from dairy cattle with
papillomatous digital dermatitis and interdigital dermatitis", veterinary
microbiology 47:343-355.
Walker, R.L., et al. (1997) "Humoral response to dairy cattle to
spirochetes isolated from papillomatous digital dermatitis lesions", AJVR
58(7):744-748.
|
Primary Examiner: Chin; Christopher L.
Assistant Examiner: Ryan; V.
Attorney, Agent or Firm: Townsend and Townsend and Crew LLP
Goverment Interests
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with Government support under Grant No. CALV AH
144, awarded by the United States Department of Agriculture. The
Government has certain rights in this invention.
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is CIP of U.S. Ser. No. 08/943,571, filed Oct. 3, 1997,
herein incorporated by reference in its entirety.
Claims
What is claimed is:
1. A biologically pure culture of ungulate Treponema, having all the
characteristics of Treponema strain 9-3379 (ATCC Patent Deposit
Designation PTA-171).
2. The culture of claim 1, which is Treponema strain 9-3379 (ATCC Patent
Deposit Designation PTA-171).
3. A pharmaceutical composition comprising a pharmaceutically acceptable
carrier and an immunogenically effective amount of an ungulate Treponema
culture of claim 1.
4. The composition of claim 3, further comprising an antigen from an
organism that causes ungulate foot rot selected from the group consisting
of Fusobacterium necrophorum, Porphyromonas levii, and Dichelobacter
nodosus.
5. The composition of claim 3, further comprising a bovine respiratory
syncytial virus antigen, a bovine Herpes virus antigen, a leptospiral
antigen, a bovine diarhhea virus antigen, a bovine parainfluenza virus
antigen, a vesicular stomatitis virus antigen, a malignant catarrhal fever
virus antigen, a blue tongue virus antigen, a pseudorabies virus antigen,
a rabies virus antigen, a rinderpest virus antigen, or a Clostridia spp.
antigen.
6. The composition of claim 3, wherein the ungulate Treponema culture is
inactivated.
7. The composition of claim 6, wherein the ungulate Treponema culture is
inactivated by freezing, lyophilization, or chemical treatment.
8. The composition of claim 3, wherein the composition is formulated for
parenteral administration.
Description
FIELD OF THE INVENTION
This invention relates to the diagnosis and prevention of ungulate diseases
caused by treponeme spirochete bacteria. The invention specifically
relates to isolated cultures of these spirochetes and their isolated
nucleic acids and proteins.
BACKGROUND OF THE INVENTION
Over the past few years, there has been a marked increase in the prevalence
of related painful diseases of the feet of dairy cattle called
papillomatous digital dermatitis (PDD), digital dermatitis (DD) or
interdigital dermatitis (IDD), hereinafter referred to as PDD. Commonly
known as footwarts, PDD has been reported in the USA, Canada, Europe, the
Mediterranean, Japan, South Africa, Australia, and South America. This
disease adversely affects the dairy industry economically through
increased treatment costs and by its negative effect on milk production
and reproductive performance. It appears as a contagious disease, with
some herds having a 90% prevalence of clinical disease.
PDD causes severe lameness, decrease in body condition, and decreased
reproductive performance in cattle. First calf heifers are most often
affected. Little or no digital swelling occurs. The lesions are limited to
the feet, usually the hind feet. Typically, lesions occur at the back of
foot near the interditigal ridge. Lesions may range from small,
dime-sized, flat, red and circumscribed lesions (early lesions) to large,
raised, golf-ball sized, with long brown/black papillary fronds (mature
lesions). The long (true) hairs at edge of lesion are frequently
hypertrophied. The lesions may persist for many months or may regress in
dry weather.
Various attempts to demonstrate viruses, structural group antigens of
papillomavirus, and bovine papillomavirus types 1-6 in PDD have been
negative (Basset et al., Vet. Rec. 126:164-165 (1990); Read et al., Vet.
Rec. 130:59-60 (1992); Read et al., Proc. Amer. Ass. Vet. Lab. Diag. 38:68
(1995); Rebhun, et al., J. Am. Vet. Med. Assoc. 177:437-440 (1980); Scavia
et al., Proc. Int. Sym. Dis. Rum. Digit 8:174-176 (1994); Zemljic, Proc.
Int. Sym. Rum. Digit 8:164-167 (1994)). Histologic examination for
Dermatophilus spp., fungi, and parasites also have been negative (Read et
al., Proc. Amer. Ass. Vet. Lab. Diag. 38:68 (1995)).
Because the disease responds to topical or parenteral treatment with
antibiotics, a bacterial role in the disease process has been indicated.
Spirochetes have been demonstrated invading into the stratum spinosum and
dermal papillae of PDD lesions and are the predominant bacterial
morphotype present. Spirochetes with morphologic, phenotypic, and genetic
characteristics of the genus Treponema have been isolated from PDD lesions
(Walker et al., Vet. Micro. 47:343-355 (1995); Walker et al., AJVR
58:744-748 (1997)). Intralesional invasive spirochetes have also been
demonstrated in PDD worldwide (Blowey et al., Vet. Rec. 135, 115-117
(1994); Scavia et al., Proc. Int. Sym. Dis. Rum. Digit 8:174-176 (1994);
Zemljic, Proc. Int. Sym. Rum. Digit 8:164-167 (1994); Kimura et al., J.
Vet. Med. Jpn. 46:899-906 (1993); Dopfer et al., Vet. Rec. 140:620-623
(1997); Choi et al., Int. J. Syst. Bact. 47:175-181 (1997); Rijpkema et
al., Vet. Rec. 140:257-259 (1997)).
It remains unclear whether these spirochete organisms have a primary role
in lesion development or whether they act as secondary opportunists after
the initial PDD lesion has developed. For example, other bacteria such as
Serpens spp., a gram negative rod related to members of the genus
Pseudomonas, have also been suggested as a PDD agent. In the field, PDD
appears contagious but most previous attempts to transmit PDD
experimentally have not been successful (Weaver, Proc. 7th Biann. Int.
Sym. Dis. Rum. Digit, Copenhagen (1992); Basset et al., Vet. Rec.
126:164-165 (1990); but see Read & Walker, Vet. Pathology 33:607 (1996)).
Currently, the etiologic agent of PDD is unknown. In addition, it is
unknown whether PDD can spread to other species, although similar
histopathologic lesions have been observed in sheep, horses, and goats.
There is therefore a need to definitively identify the etiologic agent for
PDD and to develop a means of preventing this disease by developing a
vaccine against PDD.
SUMMARY OF THE INVENTION
The present invention identifies ungulate Treponema spp. as the etiologic
agents of ungulate papillomatous digital dermatitis (PDD). The invention
therefore provides isolated cultures of Treponema spp., vaccines that
effectively immunize susceptible ungulates against PDD, and methods of
diagnosing PDD by detecting infection with Treponema spp.
In one aspect, the invention provides a biologically pure culture of
ungulate Treponema.
In one embodiment, the culture has all the characteristics of Treponema
strain 1-9185MED (ATCC Accession No. 202030), Treponema strain 2-1498
(ATCC Accession No. 202031), or Treponema strain 9-5379 (ATCC Patent
Deposit Designation PTA-171). In another embodiment, the culture is
selected from the group consisting of Treponema strain 1-9185MED (ATCC
Accession No. 202030), Treponema strain 2-1498 (ATCC Accession No.
202031), or Treponema strain 9-3379 (ATCC Patent Deposit Designation
PTA-171).
In another aspect, the invention provides a pharmaceutical composition
comprising a pharmaceutically acceptable carrier and an immunogenically
effective amount of an ungulate Treponema antigen.
In another aspect, the invention provides a method for inducing an immune
response against ungulate Treponema. This method includes the step of
administering to an ungulate animal a pharmaceutical composition
comprising a pharmaceutically acceptable carrier and an immunogenically
effective amount of an ungulate Treponema antigen.
In one embodiment, the composition further includes an antigen from an
organism that causes ungulate foot rot selected from the group consisting
of Fusobacterium necrophorum, Porphyromonas levii, and Dichelobacter
nodosus. In another embodiment, the pharmaceutical composition further
comprises a bovine respiratory syncytial virus antigen, a bovine Herpes
virus antigen, a leptospiral antigen, a bovine diarrhea virus antigen, a
bovine parainfluenza virus antigen, a vesicular stomatitis virus antigen,
a malignant catarrhal fever virus antigen, a blue tongue virus antigen, a
pseudorabies virus antigen, a rabies virus antigen, a rinderpest virus
antigen, a Fusobacterium necrophorum antigen, a Dichelobacter nodosus
antigen, or a Clostridia spp. antigen.
In another embodiment, the ungulate Treponema antigen is from Treponema
strain 1-9185MED (ATCC Accession No. 202030), Treponema strain 2-1498
(ATCC Accession No. 202031), or Treponema strain 9-3379 (ATCC Patent
Deposit Designation PTA-171). In another embodiment, the antigen is a
biologically pure culture of Treponema. In another embodiment, the culture
is selected from the group consisting of Treponema strain 1-9185MED (ATCC
Accession No. 202030), Treponema strain 2-1498 (ATCC Accession No.
202031), or Treponema strain 9-3379 (ATCC Accession Patent Deposit
Designation PTA-171). In another embodiment, the ungulate Treponema
antigen is an isolated Treponema polypeptide. In another embodiment, the
polypeptide is recombinantly produced.
In another embodiment, the pharmaceutical composition is administered
parenterally.
In another aspect, the invention provides a method of detecting the
presence of antibodies specifically immunoreactive with an ungulate
Treponema antigen in a biological sample. This method includes the steps
of contacting the sample with the Treponema antigen, thereby forming a
antigen/antibody complex; and detecting the presence or absence of the
complex.
In one embodiment, the Treponema antigen is from Treponema strain 1-9185MED
(ATCC Accession No. 202030), Treponema strain 2-1498 (ATCC Accession No.
202031), or Treponema strain 9-3379 (ATCC Patent Deposit Designation
PTA-171). In another embodiment, the biological sample is bovine serum. In
another embodiment, the antigen is an isolated Treponema polypeptide. In
another embodiment, the antigen is immobilized on a solid surface. In
another embodiment, the complex is detected using a labeled anti-bovine
antibody.
In another aspect, the invention provides a method of detecting the
presence of ungulate Treponema in a biological sample. This method
includes the steps of contacting the sample with an antibody specifically
immunoreactive with a Treponema antigen, thereby forming a
antigen/antibody complex; and detecting the presence or absence of the
complex.
In one embodiment, the antibody is specifically immunoreactive with an
antigen from Treponema strain 1-9185MED (ATCC Accession No. 202030),
Treponema strain 2-1498 (ATCC Accession No. 202031), or Treponema strain
9-3379 (ATCC Patent Deposit Designation PTA-171). In another embodiment,
the antibody is a monoclonal antibody. In another embodiment, the antibody
is immobilized on a solid surface. In another embodiment, the complex is
detected using a second labeled antibody. In another embodiment, the
biological sample is ungulate foot tissue.
In another aspect, the invention provides a method of detecting the
presence of ungulate Treponema-specific nucleic acids in a biological
sample. This method includes the steps of: contacting the sample with a
oligonucleotide probe which specifically hybridizes with a target
Treponema-specific polynucleotide sequence, thereby forming a
hybridization complex; and detecting the presence or absence of the
complex.
In one embodiment, the target Treponema-specific polynucleotide sequence is
from Treponema strain 1-9185MED (ATCC Accession No. 202030), Treponema
strain 2-1498 (ATCC Accession No. 202031), or Treponema strain 9-3379
(ATCC Patent Deposit Designation PTA-171). In another embodiment, the
target Treponema-specific polynucleotide sequence is 16S rRNA. In another
embodiment, the target Treponema-specific polynucleotide sequence is SEQ
ID NO:1 (16S rRNA from strain 2-1498), SEQ ID NO:2 (16S rRNA from strain
1-9185MED), SEQ ID NO:3 (16S rRNA from strain 7-2009), SEQ ID NO:4 (16S
rRNA from strain 9-3301), SEQ ID NO:5 (16S rRNA from strain 9-3143), SEQ
ID NO:6 (16S rRNA from strain 9-3528) SEQ ID NO:7 (16S rRNA from strain
9-3379), or SEQ ID NO:8 (16S rRNA from strain 9-227). In another
embodiment, the step of detecting further comprises amplifying the target
Treponema-specific polynucleotide sequence.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the phylogenetic relationship between the Treponema
isolates of the present application and other known Treponema species.
DETAILED DESCRIPTION OF THE INVENTION
I. Introduction
The present invention provides isolated Treponema cultures isolated from
cattle: Treponema strain 1-9185MED (ATCC Accession No. 202030), Treponema
strain 2-1498 (ATCC Accession No. 202031), or Treponema strain 9-3379
(ATCC Patent Deposit Designation PTA-171). The cultures are useful in a
variety of applications, including the production of nucleic acids and
proteins for diagnostic assays for PDD and the preparation of immunogenic
proteins and compositions for use in PDD vaccine compositions. The
cultures fall into three distinct, related groups or types of Treponema
(see FIG. 1). Group 1 comprises strains 2-1498, 7-2009, and 9-3301. Group
2 comprises strains 1-9185MED, 9-3143, and 9-3528. Group 3 comprises
strains 9-3379 and 9-27. Each of these strains is valuable for generating
PDD vaccines against individual strains of Treponema. In addition, a
vaccine against a particular group of Treponema can also be generated. The
three different Treponema groups described herein may be of used to
prepare vaccines for different geographic regions, where one group of
Treponema may be the dominant group causing PDD.
II. Definitions
As used herein, the following terms have the meanings ascribed to them
unless specified otherwise.
A "biologically pure culture" refers to a continuous in vitro culture of
ungulate Treponema which is substantially free of other organisms. A
culture is substantially free of other organisms if standard harvesting
procedures (as described below) result in a preparation which comprises at
least about 95%, preferably 99% or more of the organism, e.g., Treponema.
"Ungulate Treponema" and "bovine Treponema" refer to flexible,
spiral-shaped spirochete bacteria of the Treponema genus identified in or
isolated from ungulate and bovine biological samples, in particular from
hoof and foot tissue. "Ungulate" refers to hooved animals such as cows,
horses, sheep, and goats. "Bovine" refers to cattle (bulls, cows, calves).
Typically, the spirochetes of the Treponema genus can be isolated from
foot or hoof tissue of hooved animals infected with PDD. Exemplary
Treponema isolates include Treponema strain 1-9185MED (ATCC Accession No.
202030), Treponema strain 2-1498 (ATCC Accession No. 202031), or Treponema
strain 9-3379 (ATCC Patent Deposit Designation PTA-171).
"Biological sample" refers to any sample obtained from a living or dead
organism. Examples of biological samples include biological fluids and
tissue specimens. Examples of tissue specimens include bovine hoof and
foot tissue. Such biological samples can be prepared for analysis using in
situ techniques.
"Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and
polymers thereof in either single- or double-stranded form. The term
encompasses nucleic acids containing known nucleotide analogs or modified
backbone residues or linkages, which are synthetic, naturally occurring,
and non-naturally occurring, which have similar binding properties as the
reference nucleic acid, and which are metabolized in a manner similar to
the reference nucleotides. Examples of such analogs include, without
limitation, phosphorothioates, phosphoramidates, methyl phosphonates,
chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic
acids (PNAs).
Unless otherwise indicated, a particular nucleic acid sequence also
implicitly encompasses conservatively modified variants thereof (e.g.,
degenerate codon substitutions) and complementary sequences, as well as
the sequence explicitly indicated. The term nucleic acid is used
interchangeably with gene, cDNA, mRNA, oligonucleotide, and
polynucleotide.
The terms "polypeptide," "peptide" and "protein" are used interchangeably
herein to refer to a polymer of amino acid residues. The terms apply to
amino acid polymers in which one or more amino acid residue is an analog
or mimetic of a corresponding naturally occurring amino acid, as well as
to naturally occurring amino acid polymers.
The term "amino acid" refers to naturally occurring and synthetic amino
acids, as well as amino acid analogs and amino acid mimetics that function
in a manner similar to the naturally occurring amino acids. Naturally
occurring amino acids are those encoded by the genetic code, as well as
those amino acids that are later modified, e.g., hydroxyproline,
.gamma.-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers
to compounds that have the same basic chemical structure as a naturally
occurring amino acid, i.e., an .alpha. carbon that is bound to a hydrogen,
a carboxyl group, an amino group, and an R group., e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified peptide
backbones, but retain the same basic chemical structure as a naturally
occurring amino acid. Amino acid mimetics refers to chemical compounds
that have a structure that is different from the general chemical
structure of an amino acid, but that functions in a manner similar to a
naturally occurring amino acid.
Amino acids may be referred to herein by either their commonly known three
letter symbols or by the one-letter symbols recommended by the IUPAC-IUB
Biochemical Nomenclature Commission. Nucleotides, likewise, may be
referred to by their commonly accepted single-letter codes.
"Conservatively modified variants" applies to both amino acid and nucleic
acid sequences. With respect to particular nucleic acid sequences,
conservatively modified variants refers to those nucleic acids which
encode identical or essentially identical amino acid sequences, or where
the nucleic acid does not encode an amino acid sequence, to essentially
identical sequences. Specifically, degenerate codon substitutions may be
achieved by generating sequences in which the third position of one or
more selected (or all) codons is substituted with mixed-base and/or
deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);
Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al.,
Mol. Cell. Probes 8:91-98 (1994)). Because of the degeneracy of the
genetic code, a large number of functionally identical nucleic acids
encode any given protein. For instance, the codons GCA, GCC, GCG and GCU
all encode the amino acid alanine. Thus, at every position where an
alanine is specified by a codon, the codon can be altered to any of the
corresponding codons described without altering the encoded polypeptide.
Such nucleic acid variations are "silent variations," which are one
species of conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible silent
variation of the nucleic acid. One of skill will recognize that each codon
in a nucleic acid (except AUG, which is ordinarily the only codon for
methionine, and TGG, which is ordinarily the only codon for tryptophan)
can be modified to yield a functionally identical molecule. Accordingly,
each silent variation of a nucleic acid which encodes a polypeptide is
implicit in each described sequence.
As to amino acid sequences, one of skill will recognize that individual
substitutions, deletions or additions to a nucleic acid, peptide,
polypeptide, or protein sequence which alters, adds or deletes a single
amino acid or a small percentage of amino acids in the encoded sequence is
a "conservatively modified variant" where the alteration results in the
substitution of an amino acid with a chemically similar amino acid.
Conservative substitution tables providing functionally similar amino
acids are well known in the art. Such conservatively modified variants are
in addition to and do not exclude polymorphic variants, interspecies
homologs, and alleles of the invention.
The following groups each contain amino acids that are conservative
substitutions for one another:
1) Alanine (A), Glycine (G);
2) Serine (S), Threonine (T);
3) Aspartic acid (D), Glutamic acid (E);
4) Asparagine (N), Glutamine (Q);
5) Cysteine (C), Methionine (M);
6) Arginine (R), Lysine (K), Histidine (H);
7) Isoleucine (I), Leucine (L), Valine (V); and
8) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
(see, e.g., Creighton, Proteins (1984)).
The terms "identical" or percent "identity," in the context of two or more
nucleic acids or polypeptide sequences, refer to two or more sequences or
subsequences that are the same or have a specified percentage of amino
acid residues or nucleotides that are the same, when compared and aligned
for maximum correspondence over a comparison window, as measured using one
of the following sequence comparison algorithms or by manual alignment and
visual inspection. This definition also refers to the complement of a test
sequence, which has a designated percent sequence or subsequence
complementarity when the test sequence has a designated or substantial
identity to a reference sequence. For example, a designated amino acid
percent identity of 95% refers to sequences or subsequences that have at
least about 95% amino acid identity when aligned for maximum
correspondence over a comparison window as measured using one of the
following sequence comparison algorithms or by manual alignment and visual
inspection. Such sequences would then be said to have substantial
identity, or to be substantially identical to each other. Preferably,
sequences have at least about 70% identity, more preferably 80% identity,
more preferably 90-95% identity and above. Preferably, the percent
identity exists over a region of the sequence that is at least about 25
amino acids in length, more preferably over a region that is 50-100 amino
acids in length.
When percentage of sequence identity is used in reference to proteins or
peptides, it is recognized that residue positions that are not identical
often differ by conservative amino acid substitutions, where amino acids
residues are substituted for other amino acid residues with similar
chemical properties (e.g., charge or hydrophobicity) and therefore do not
change the functional properties of the molecule. Where sequences differ
in conservative substitutions, the percent sequence identity may be
adjusted upwards to correct for the conservative nature of the
substitution. Means for making this adjustment are well known to those of
skill in the art. Typically this involves scoring a conservative
substitution as a partial rather than a full mismatch, thereby increasing
the percentage sequence identity. Thus, for example, where an identical
amino acid is given a score of 1 and a non-conservative substitution is
given a score of zero, a conservative substitution is given a score
between zero and 1. The scoring of conservative substitutions is
calculated according to, e.g., the algorithm of Meyers & Miller, Computer
Applic. Biol. Sci. 4:11-17 (1988) e.g., as implemented in the program
PC/GENE (Intelligenetics, Mountain View, Calif., USA).
For sequence comparison, typically one sequence acts as a reference
sequence, to which test sequences are compared. When using a sequence
comparison algorithm, test and reference sequences are entered into a
computer, subsequence coordinates are designated, if necessary, and
sequence algorithm program parameters are designated. Default program
parameters can be used, or alternative parameters can be designated. The
sequence comparison algorithm then calculates the percent sequence
identity for the test sequence(s) relative to the reference sequence,
based on the designated or default program parameters.
A "comparison window", as used herein, includes reference to a segment of
any one of the number of contiguous positions selected from the group
consisting of from 25 to 600, usually about 50 to about 200, more usually
about 100 to about 150 in which a sequence may be compared to a reference
sequence of the same number of contiguous positions after the two
sequences are optimally aligned. Methods of alignment of sequences for
comparison are well-known in the art. Optimal alignment of sequences for
comparison can be conducted, e.g., by the local homology algorithm of
Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the
search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci.
USA 85:2444 (1988), by computerized implementations of these algorithms
(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software
Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by
manual alignment and visual inspection (see, e.g., Ausubel et al., supra).
One example of a useful algorithm is PILEUP. PILEUP creates a multiple
sequence alignment from a group of related sequences using progressive,
pairwise alignments to show relationship and percent sequence identity. It
also plots a tree or dendogram showing the clustering relationships used
to create the alignment. PILEUP uses a simplification of the progressive
alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987). The
method used is similar to the method described by Higgins & Sharp, CABIOS
5:151-153 (1989). The program can align up to 300 sequences, each of a
maximum length of 5,000 nucleotides or amino acids. The multiple alignment
procedure begins with the pairwise alignment of the two most similar
sequences, producing a cluster of two aligned sequences. This cluster is
then aligned to the next most related sequence or cluster of aligned
sequences. Two clusters of sequences are aligned by a simple extension of
the pairwise alignment of two individual sequences. The final alignment is
achieved by a series of progressive, pairwise alignments. The program is
run by designating specific sequences and their amino acid or nucleotide
coordinates for regions of sequence comparison and by designating the
program parameters. Using PILEUP, a reference sequence is compared to
other test sequences to determine the percent sequence identity
relationship using the following parameters: default gap weight (3.00),
default gap length weight (0.10), and weighted end gaps. PILEUP can be
obtained from the GCG sequence analysis software package, e.g, version 7.0
(Devereaux et al., Nuc. Acids Res. 12:387-395 (1984).
Another example of algorithm that is suitable for determining percent
sequence identity (i.e., substantial similarity or identity) is the BLAST
algorithm, which is described in Altschul et al., J. Mol. Biol.
215:403-410 (1990). Software for performing BLAST analyses is publicly
available through the National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying
high scoring sequence pairs (HSPs) by identifying short words of length W
in the query sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold (Altschul et al, supra). These initial neighborhood word hits
act as seeds for initiating searches to find longer HSPs containing them.
The word hits are then extended in both directions along each sequence for
as far as the cumulative alignment score can be increased. Cumulative
scores are calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N (penalty
score for mismatching residues, always <0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension of the
word hits in each direction are halted when: the cumulative alignment
score falls off by the quantity X from its maximum achieved value; the
cumulative score goes to zero or below, due to the accumulation of one or
more negative-scoring residue alignments; or the end of either sequence is
reached. The BLAST algorithm parameters W, T, and X determine the
sensitivity and speed of the alignment. The BLASTN program (for nucleotide
sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of
10, M=5, N=4, and a comparison of both strands. For amino acid sequences,
the BLASTP program uses as default parameters a wordlength (W) of 3, an
expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff &
Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
The BLAST algorithm also performs a statistical analysis of the similarity
between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad.
Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the
BLAST algorithm is the smallest sum probability (P(N)), which provides an
indication of the probability by which a match between two nucleotide or
amino acid sequences would occur by chance. For example, a nucleic acid is
considered similar to a reference sequence if the smallest sum probability
in a comparison of the test nucleic acid to the reference nucleic acid is
less than about 0.1, more preferably less than about 0.01, and most
preferably less than about 0.001.
An indication that two nucleic acid sequences or polypeptides are
substantially identical is that the polypeptide encoded by the first
nucleic acid is immunologically cross reactive with the antibodies raised
against the polypeptide encoded by the second nucleic acid, as described
below. Thus, a polypeptide is typically substantially identical to a
second polypeptide, for example, where the two peptides differ only by
conservative substitutions. Another indication that two nucleic acid
sequences are substantially identical is that the two molecules or their
complements hybridize to each other under stringent conditions, as
described below.
Another indication that polynucleotide sequences are substantially
identical is if two molecules hybridize to each other under stringent
conditions. Stringent conditions are sequence dependent and will be
different in different circumstances. Generally, stringent conditions are
selected to be about 5.degree. C. lower than the thermal melting point
(Tm) for the specific sequence at a defined ionic strength and pH. The Tm
is the temperature (under defined ionic strength and pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. Typically
stringent conditions for a Southern blot protocol involve hybridizing in a
buffer comprising 5.times. SSC, 1% SDS at 65.degree. C. or hybridizing in
a buffer containing 5.times. SSC and 1% SDS at 42.degree. C. and washing
at 65.degree. C. with a 0.2.times. SSC, 0.1% SDS wash.
The phrase "specifically or selectively hybridizing to," refers to
hybridization between a probe and a target sequence in which the probe
binds substantially only to the target sequence, forming a hybridization
complex, when the target is in a heterogeneous mixture of polynucleotides
and other compounds. Such hybridization is determinative of the presence
of the target sequence. Although the probe may bind other unrelated
sequences, at least 90%, preferably 95% or more of the hybridization
complexes formed are with the target sequence.
"Antibody" refers to an immunoglobulin molecule able to bind to a specific
epitope on an antigen. Antibodies can be a polyclonal mixture or
monoclonal. Antibodies can be intact immunoglobulins derived from natural
sources or from recombinant sources and can be immunoreactive portions of
intact immunoglobulins. Antibodies may exist in a variety of forms
including, for example, Fv, F.sub.ab, and F(ab).sub.2, as well as in
single chains. Single-chain antibodies, in which genes for a heavy chain
and a light chain are combined into a single coding sequence, may also be
used.
An "antigen" is a molecule that is recognized and bound by an antibody,
e.g., peptides, carbohydrates, organic molecules, or more complex
molecules such as glycolipids and glycoproteins. The part of the antigen
that is the target of antibody binding is an antigenic determinant and a
small functional group that corresponds to a single antigenic determinant
is called a hapten.
The phrase "specifically immunoreactive with", when referring to a protein
or peptide, refers to a binding reaction between the protein and an
antibody which is determinative of the presence of the protein in the
presence of a heterogeneous population of proteins and other compounds.
Thus, under designated immunoassay conditions, the specified antibodies
bind to a particular protein and do not bind in a significant amount to
other proteins present in the sample. Specific binding to an antibody
under such conditions may require an antibody that is selected for its
specificity for a particular protein. A variety of immunoassay formats may
be used to select antibodies specifically immunoreactive with a particular
protein and are described in detail below.
The phrase "substantially pure" or "isolated" when referring to a Treponema
peptide or protein, means a chemical composition which is free of other
subcellular components of the Treponema organism. Typically, a monomeric
protein is substantially pure when at least about 85% or more of a sample
exhibits a single polypeptide backbone. Minor variants or chemical
modifications may typically share the same polypeptide sequence. Depending
on the purification procedure, purities of 85%, and preferably over 95%
pure are possible. Protein purity or homogeneity may be indicated by a
number of means well known in the art, such as polyacrylamide gel
electrophoresis of a protein sample, followed by visualizing a single
polypeptide band on a polyacrylamide gel upon silver staining. For certain
purposes high resolution will be needed and HPLC or a similar means for
purification utilized.
A "label" is a composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, or chemical means. For example, useful labels
include .sup.32 P, fluorescent dyes, electron-dense reagents, enzymes
(e.g., as commonly used in an ELISA), biotin, dioxigenin, or haptens and
proteins for which antisera or monoclonal antibodies are available.
As used herein a "nucleic acid probe or oligonucleotide" is defined as a
nucleic acid capable of binding to a target nucleic acid of complementary
sequence through one or more types of chemical bonds, usually through
complementary base pairing, usually through hydrogen bond formation. As
used herein, a probe may include natural (i.e., A, G, C, or T) or modified
bases (7-deazaguanosine, inosine, etc.). In addition, the bases in a probe
may be joined by a linkage other than a phosphodiester bond, so long as it
does not interfere with hybridization. Thus, for example, probes may be
peptide nucleic acids in which the constituent bases are joined by peptide
bonds rather than phosphodiester linkages. It will be understood by one of
skill in the art that probes may bind target sequences lacking complete
complementarity with the probe sequence depending upon the stringency of
the hybridization conditions. The probes are preferably directly labeled
as with isotopes, chromophores, lumiphores, chromogens, or indirectly
labeled such as with biotin to which a streptavidin complex may later
bind. By assaying for the presence or absence of the probe, one can detect
the presence or absence of the select sequence or subsequence.
A "labeled nucleic acid probe or oligonucleotide" is one that is bound,
either covalently, through a linker, or through ionic, van der Waals or
hydrogen bonds to a label such that the presence of the probe may be
detected by detecting the presence of the label bound to the probe.
"Amplification" primers are oligonucleotides comprising either natural or
analogue nucleotides that can serve as the basis for the amplification of
a select nucleic acid sequence. They include, e.g., polymerase chain
reaction primers and ligase chain reaction oligonucleotides.
The term "recombinant" when used with reference to a cell, or nucleic acid,
or vector, indicates that the cell, or nucleic acid, or vector, has been
modified by the introduction of a heterologous nucleic acid or the
alteration of a native nucleic acid, or that the cell is derived from a
cell so modified. Thus, for example, recombinant cells express genes that
are not found within the native (non-recombinant) form of the cell or
express native genes that are otherwise abnormally expressed, under
expressed or not expressed at all.
"Pharmaceutically acceptable" means a material that is not biologically or
otherwise undesirable, i.e., the material can be administered to an
individual along with a Treponema antigen without causing any undesirable
biological effects or interacting in a deleterious manner with any of the
other components of the pharmaceutical composition.
III. Isolation and characterization of ungulate Treponema from PDD infected
cows
Treponema cultures of the invention have been deposited with the American
Type Culture Collection, 10801 University Boulevard, Manassas, Va.
20110-2209, on Sep. 16, 1997 and assigned Accession Numbers 202030
(1-9185MED) and 202031 (2-1498). Treponema strain 9-3379 (ATCC Deposit
Designation PTA-171) have been deposited with the American Type Culture
Collection, 10801 University Boulevard, Manassas, Va. 20110-2209, on Jun.
4, 1999.
Treponema isolates that cause PDD are typically obtained from bovine foot
tissue of cows infected with PDD (see, e.g., Walker et al., Vet. Micro.
47:343-355 (1995)). These cultures are generally inoculated with tissue
from foot biopsies or scraping of PDD lesions in affected cow foot tissue.
Isolates are grown in suitable bacterial media, preferably oral treponome
isolation (OTI) broth or agar. Pure isolates are obtained by plating out
cultures, picking individual colonies, propagating the colonies and
re-plating to assess purity.
Generally, morphological, histopathological, and immunological
characteristics are used to identify isolated Treponema spirochetes
causing lesions in the foot tissue of these cows with PDD.
Stereomicroscope studies are used to characterize the morphological
features of isolates. Distinguishing histopathological and morphological
features include active flexing, rotational motility, number of axial
filaments, cell diameter, cell length, and wavelength. Enzyme activity of
typical enzymes such as alkaline phosphatase, esterase, esterase lipase,
acid phosphatase, naphthol-AS-BI-phosphohydrolase, .beta.-galactosidase,
.beta.-glucuronidase, and N-acetyl-.beta.-glucosaminidase are evaluated,
typically using a commercially available system (e.g., API ZYM, Bio
Merieux, France. Restriction enzyme analysis on genomic DNA is performed
according to standard methodology. Standard immunoblot analysis is used to
examine proteins and other antigens. Antigenically, antigens from isolates
generally fell into three groups, those that react strongly with 1-9185MED
antisera, those that react with 2-1498 antisera, and those that react
strongly with 9-3379 antisera. A monoclonal antibody to a Borrelia
burgdorferi antigen is used as a negative control (another spirochete
genus). Based on morphological, histopathological, and antigenic
characteristics, Treponema spirochetes can be distinguished from the other
seven genera of spirochetes, Borellia, Brachyspira, Cristispira,
Leptonema, Leptospira, Serpulina, and Spirochaeta (Holt et al., in Bergy's
Manual of Determinative Bacteriology, (9th ed. 1994)).
In addition, 16S rRNA sequences for eight bovine Treponema isolates were
obtained and compared using PCR to the 16S rRNA of other bacteria,
including other spirochetes. PCR reactions were performed according to
standard methods known to those skilled in the art. Alignment of these
sequences with published sequences of Treponema phagedenis, T. pallidum,
T. pectinovorum, T. denticola, T. saccharophilum, T. bryantii, T.
vicentii, and Escherichia coli showed that the bovine Treponema isolates
are genotypically unique. For example, the 16S rRNA sequences from
Treponema strain 1-9185MED (SEQ ID NO:2) most closely aligns with T.
denticola, 2-1498 (SEQ ID NO: 1) most closely aligns with T. phagedenis,
and 9-3379 (SEQ ID NO: 7) aligns most closely with T. vincentii, but the
sequences do not show 100% homology (see also FIG. 1). The 16S rRNA from
bovine Treponema is therefore useful as a diagnostic tool for PDD. To
choose suitable PCR primers for use in diagnostic assays, the 16S rRNAs of
the most closely related bacterial species are aligned with the 16S rRNA
of each Treponema strains (SEQ ID NOS:1-8). The sequence alignment allows
selection of primers that are used to specifically amplify Treponema
strains 1-9185MED, 2-1498, 7-2009, 9-3301, 9-3143, 9-3528, 9-3379, and
9-227. For example, the following primers were used to examine and
generate the 16S rRNA of strains 1-9185MED and 2-1498 (SEQ ID NOS:1 and
2). The letters "R" and "F" designate reverse and forward respectively.
All primers begin with the 5' end (For E. coli primer sequences and
primers 11 and 12, see Eden et al., Int. J. Systematic Bacteriol., pp.
324-325 (1991)).
1. F-(E. coli base 8 to 27) AGAGTTTGATCCTGGCTCAG (SEQ ID NO:9)
2. R-(E. coli complement bases 1510 to 1492) ggttaccttgttacgactt (SEQ ID
NO:10)
3. F-TGCTTGAGGATGAGCCC (SEQ ID NO:11)
4. R-AAGCAAGGTCGTAGGCTCC (SEQ ID NO:12)
5. F-AAGAATAAGGAGATGAGGG (SEQ ID NO:13)
6. F-ATGAGGGAATGCGTCCTTG (SEQ ID NO:14)
7. F-AAGGGTGAAACTCAAAGG (SEQ ID NO15)
8. F-CAGGATCAAACTCTATTGGG (SEQ ID NO:16)
9. R-TTCACCCTTGCGGGCATACT (SEQ ID NO:17)
10. F-ATTACGTGCCAGCAGCCGCG (SEQ ID NO:18)
11. R-CTGCTGCCTCCCGTA (SEQ ID NO:19)
12. R-CGTATTACCGCGGCTGCT (SEQ ID NO:20)
ISOLATE PRIMER
1-9185MED 5, 7, 11, 4, 12, 9
2-1498 2, 3, 11, 4
As explained in detail below, the isolates are useful in a variety of
diagnostic assays as well as pharmaceutical compositions for treatment and
prevention of PDD.
IV. Preparation of Treponema polypeptides and nucleic acids
Standard protein isolation and purification techniques can be used to
isolate proteins from the cultures provided here. Such techniques include
standard immunoblot techniques, selective precipitation with such
substances as ammonium sulfate, column chromatography, immunopurification
methods, and the like (see, e.g., Scopes, Protein Purification: Principles
and Practice (1982)).
Once proteins have been identified, standard protein purification methods
can be used to purify these proteins and produce polyclonal or monoclonal
antibodies for use in diagnostic methods described below. Such antigens
are useful in enzyme-linked immunoassays (ELISA) and immunoperoxidase
assays on in situ fixed tissue for the detection of Treponema-specific
antibodies in PDD infected cattle.
Rather than extract the proteins directly from cultured Treponema, nucleic
acids derived from the cultures can be used for recombinant expression of
the proteins. In these methods, the nucleic acids encoding the proteins of
interest are introduced into suitable host cells, followed by induction of
the cells to produce large amounts of the protein. The invention relies on
routine techniques in the field of recombinant genetics, well known to
those of ordinary skill in the art. A basic text disclosing the general
methods of use in this invention is Sambrook et al., Molecular Cloning, A
Laboratory Manual (2nd ed. 1989).
Nucleic acids for use as diagnostic oligonucleotide probes or for the
recombinant expression of proteins can be isolated using a number of
techniques. For instance, portions of proteins isolated from the cultures
discussed above can be sequenced and used to design degenerate
oligonucleotide probes to screen a cDNA library. Amino acid sequencing is
performed and oligonucleotide probes are synthesized according to standard
techniques as described, for instance, in Sambrook et al., supra.
Alternatively, oligonucleotide probes useful for identification of desired
genes can also be prepared from conserved regions of related genes in
other species.
Alternatively, amplification techniques such as polymerase chain reaction
technology (PCR) can be used to amplify nucleic acid sequences of the
desired gene directly from mRNA, from cDNA, from genomic libraries or cDNA
libraries. Polymerase chain reaction (PCR) or other in vitro amplification
methods may also be useful, for example, to clone nucleic acid sequences
that code for proteins to be expressed, to make nucleic acids to use as
probes for detecting the presence of the mRNA in physiological samples,
for nucleic acid sequencing, or for other purposes (for a general overview
of PCR, see PCR Protocols: A Guide to Methods and Applications. (Innis et
al., eds., 1990).
Standard transfection methods are used to produce prokaryotic, mammalian,
yeast or insect cell lines which express large quantities of the desired
polypeptide, which is then purified using standard techniques (see, e.g.,
Colley et al., J. Biol. Chem. 264:17619-17622, 1989; Guide to Protein
Purification, supra).
The nucleotide sequences used to transfect the host cells can be modified
to yield Treponema polypeptides with a variety of desired properties. For
example, the polypeptides can vary from the naturally-occurring sequence
at the primary structure level by amino acid, insertions, substitutions,
deletions, and the like. These modifications can be used in a number of
combinations to produce the final modified protein chain.
The amino acid sequence variants can be prepared with various objectives in
mind, including facilitating purification and preparation of the
recombinant polypeptide. The modified polypeptides are also useful for
modifying plasma half life, improving therapeutic efficacy, and lessening
the severity or occurrence of side effects during therapeutic use. The
amino acid sequence variants are usually predetermined variants not found
in nature but exhibit the same immunogenic activity as naturally occurring
protein. In general, modifications of the sequences encoding the
polypeptides may be readily accomplished by a variety of well-known
techniques, such as site-directed mutagenesis (see Gillman & Smith, Gene
8:81-97 (1979); Roberts et al., Nature 328:731-734 (1987)). One of
ordinary skill will appreciate that the effect of many mutations is
difficult to predict. Thus, most modifications are evaluated by routine
screening in a suitable assay for the desired characteristic. For
instance, the effect of various modifications on the ability of the
polypeptide to elicit a protective immune response can be easily
determined using in vitro assays. For instance, the polypeptides can be
tested for their ability to induce lymphoproliferation, T cell
cytotoxicity, or cytokine production using standard techniques.
The particular procedure used to introduce the genetic material into the
host cell for expression of the polypeptide is not particularly critical.
Any of the well known procedures for introducing foreign nucleotide
sequences into host cells may be used. These include the use of calcium
phosphate transfection, spheroplasts, electroporation, liposomes,
microinjection, plasmid vectors, viral vectors and any of the other well
known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or
other foreign genetic material into a host cell (see Sambrook et al.,
supra). It is only necessary that the particular procedure utilized be
capable of successfully introducing at least one gene into the host cell
which is capable of expressing the gene.
Any of a number of well known cells and cell lines can be used to express
the polypeptides of the invention. For instance, prokaryotic cells such as
E. coli can be used. Eukaryotic cells include, yeast, Chinese hamster
ovary (CHO) cells, COS cells, and insect cells.
The particular vector used to transport the genetic information into the
cell is also not particularly critical. Any of the conventional vectors
used for expression of recombinant proteins in prokaryotic and eukaryotic
cells may be used. Expression vectors for mammalian cells typically
contain regulatory elements from eukaryotic viruses.
The expression vector typically contains a transcription unit or expression
cassette that contains all the elements required for the expression of the
polypeptide DNA in the host cells. A typical expression cassette contains
a promoter operably linked to the DNA sequence encoding a polypeptide and
signals required for efficient polyadenylation of the transcript. The term
"operably linked" as used herein refers to linkage of a promoter upstream
from a DNA sequence such that the promoter mediates transcription of the
DNA sequence. The promoter is preferably positioned about the same
distance from the heterologous transcription start site as it is from the
transcription start site in its natural setting. As is known in the art,
however, some variation in this distance can be accommodated without loss
of promoter function.
Following the growth of the recombinant cells and expression of the
polypeptide, the culture medium is harvested for purification of the
secreted protein. The media are typically clarified by centrifugation or
filtration to remove cells and cell debris and the proteins are
concentrated by adsorption to any suitable resin or by use of ammonium
sulfate fractionation, polyethylene glycol precipitation, or by
ultrafiltration. Other routine means known in the art may be equally
suitable. Further purification of the polypeptide can be accomplished by
standard techniques, for example, affinity chromatography, ion exchange
chromatography, sizing chromatography, His.sub.6 tagging and Ni-agarose
chromatography (as described in Dobeli et al., Mol. and Biochem. Parasit.
41:259-268 (1990)), or other protein purification techniques to obtain
homogeneity. The purified proteins are then used to produce pharmaceutical
compositions, as described below.
An alternative method of preparing recombinant polypeptides useful as
vaccines involves the use of recombinant viruses (e.g., vaccinia).
Vaccinia virus is grown in suitable cultured mammalian cells such as the
HeLa S3 spinner cells, as described by Mackett et al., in DNA cloning Vol.
II: A practical approach, pp. 191-211 (Glover, ed.).
V. Antibody Production
The isolated proteins or cultures of the present invention can be used to
produce antibodies specifically reactive with Treponema antigens. If
isolated proteins are used, they may be recombinantly produced or isolated
from Treponema cultures. Synthetic peptides made using the protein
sequences may also be used.
Methods of production of polyclonal antibodies are known to those of skill
in the art. In brief, an immunogen, preferably a purified protein, is
mixed with an adjuvant and animals are immunized. When appropriately high
titers of antibody to the immunogen are obtained, blood is collected from
the animal and antisera is prepared. Further fractionation of the antisera
to enrich for antibodies reactive to Treponema proteins can be done if
desired (see Harlow & Lane, Antibodies: A Laboratory Manual (1988)).
For example, polyclonal antisera to the 1-9185MED and 2-1498 isolates have
been produced and evaluated. The polyclonal antisera are used to identify
and characterize Treponema in the tissues of infected animals using, for
instance, in situ techniques and immunoperoxidase test procedures
described in Anderson et al. JAVMA 198:241 (1991) and Barr et al. Vet.
Pathol. 28:110-116 (1991) (see also Example 3).
Monoclonal antibodies may be obtained by various techniques familiar to
those skilled in the art. Briefly, spleen cells from an animal immunized
with a desired antigen are immortalized, commonly by fusion with a myeloma
cell (see Kohler & Milstein, Eur. J. Immunol. 6:511-519 (1976)).
Alternative methods of immortalization include transformation with Epstein
Barr Virus, oncogenes, or retroviruses, or other methods well known in the
art. Colonies arising from single immortalized cells are screened for
production of antibodies of the desired specificity and affinity for the
antigen, and yield of the monoclonal antibodies produced by such cells may
be enhanced by various techniques, including injection into the peritoneal
cavity of a vertebrate host.
Monoclonal antibodies produced in such a manner are used, for instance, in
ELISA diagnostic tests, immunoperoxidase tests, immunohistochemical tests,
for the in vitro evaluation of spirochete invasion, to select candidate
antigens for vaccine development, protein isolation, and for screening
genomic and cDNA libraries to select appropriate gene sequences.
VI. Diagnosis of Treponema infections
The present invention also provides methods for detecting the presence or
absence of Treponema in a biological sample. For instance, antibodies
specifically reactive with Treponema can be detected using either
Treponema proteins or the isolates described here. The proteins and
isolates can also be used to raise specific antibodies (either monoclonal
or polyclonal) to detect the antigen in a sample. In addition, the nucleic
acids disclosed and claimed here can be used to detect Treponema-specific
sequences using standard hybridization techniques. Each of these assays is
described below.
A. Immunoassays
For a review of immunological and immunoassay procedures in general, see
Basic and Clinical Immunology (Stites & Terr ed., 7th ed. 1991)). The
immunoassays of the present invention can be performed in any of several
configurations, which are reviewed extensively in Enzyme Immunoassay
(Maggio, ed., 1980); Tijssen, Laboratory Techniques in Biochemistry and
Molecular Biology (1985)). For instance, the proteins and antibodies
disclosed here are conveniently used in ELISA, immunoblot analysis and
agglutination assays. Particularly preferred assay formats include the
immunoperoxidase assay as described in Example 3.
In brief, immunoassays to measure anti-Treponema antibodies or antigens can
be either competitive or noncompetitive binding assays. In competitive
binding assays, the sample analyte (e.g., anti-Treponema antibodies)
competes with a labeled analyte (e.g., anti-Treponema monoclonal antibody)
for specific binding sites on a capture agent (e.g., isolated Treponema
protein) bound to a solid surface. The concentration of labeled analyte
bound to the capture agent is inversely proportional to the amount of free
analyte present in the sample.
Noncompetitive assays are typically sandwich assays, in which the sample
analyte is bound between two analyte-specific binding reagents. One of the
binding agents is used as a capture agent and is bound to a solid surface.
The second binding agent is labelled and is used to measure or detect the
resultant complex by visual or instrument means.
A number of combinations of capture agent and labelled binding agent can be
used. For instance, an isolated Treponema protein or culture can be used
as the capture agent and labelled anti-bovine antibodies specific for the
constant region of bovine antibodies can be used as the labelled binding
agent. Goat, sheep and other non-bovine antibodies specific for bovine
immunoglobulin constant regions (e.g., .gamma. or .mu.) are well known in
the art. Alternatively, the anti-bovine antibodies can be the capture
agent and the antigen can be labelled.
Various components of the assay, including the antigen, anti-Treponema
antibody, or anti-bovine antibody, may be bound to a solid surface. Many
methods for immobilizing biomolecules to a variety of solid surfaces are
known in the art. For instance, the solid surface may be a membrane (e.g.,
nitrocellulose), a microtiter dish (e.g., PVC or polystyrene) or a bead.
The desired component may be covalently bound or noncovalently attached
through nonspecific bonding.
Alternatively, the immunoassay may be carried out in liquid phase and a
variety of separation methods may be employed to separate the bound
labeled component from the unbound labelled components. These methods are
known to those of skill in the art and include immunoprecipitation, column
chromatography, adsorption, addition of magnetizable particles coated with
a binding agent and other similar procedures.
An immunoassay may also be carried out in liquid phase without a separation
procedure. Various homogeneous immunoassay methods are now being applied
to immunoassays for protein analytes. In these methods, the binding of the
binding agent to the analyte causes a change in the signal emitted by the
label, so that binding may be measured without separating the bound from
the unbound labelled component.
Western blot (immunoblot) analysis can also be used to detect the presence
of antibodies to Treponema in the sample. This technique is a reliable
method for confirming the presence of antibodies against a particular
protein in the sample. The technique generally comprises separating
proteins by gel electrophoresis on the basis of molecular weight,
transferring the separated proteins to a suitable solid support, (such as
a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and
incubating the sample with the separated proteins. This causes specific
target antibodies present in the sample to bind their respective proteins.
Target antibodies are then detected using labeled anti-bovine antibodies.
The immunoassay formats described above employ labelled assay components.
The label can be in a variety of forms. The label may be coupled directly
or indirectly to the desired component of the assay according to methods
well known in the art. A wide variety of labels may be used. The component
may be labelled by any one of several methods. Traditionally a radioactive
label incorporating .sup.3 H, .sup.125 I, .sup.35 S, .sup.14 C, or .sup.32
P was used. Non-radioactive labels include ligands which bind to labelled
antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies
which can serve as specific binding pair members for a labelled ligand.
The choice of label depends on sensitivity required, ease of conjugation
with the compound, stability requirements, and available instrumentation.
Enzymes of interest as labels will primarily be hydrolases, particularly
phosphatases, esterases and glycosidases, or oxidoreductases, particularly
peroxidases. Fluorescent compounds include fluorescein and its
derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.
Chemiluminescent compounds include luciferin, and
2,3-dihydrophthalazinediones, e.g., luminol. For a review of various
labelling or signal producing systems which may be used, see U.S. Pat. No.
4,391,904, which is incorporated herein by reference.
Non-radioactive labels are often attached by indirect means. Generally, a
ligand molecule (e.g., biotin) is covalently bound to the molecule. The
ligand then binds to an anti-ligand (e.g., streptavidin) molecule which is
either inherently detectable or covalently bound to a signal system, such
as a detectable enzyme, a fluorescent compound, or a chemiluminescent
compound. A number of ligands and anti-ligands can be used. Where a ligand
has a natural anti-ligand, for example, biotin, thyroxine, and cortisol,
it can be used in conjunction with the labelled, naturally occurring
anti-ligands. Alternatively, any haptenic or antigenic compound can be
used in combination with an antibody.
Some assay formats do not require the use of labelled components. For
instance, agglutination assays can be used to detect the presence of the
target antibodies. In this case, antigen-coated particles are agglutinated
by samples comprising the target antibodies. In this format, none of the
components need be labelled and the presence of the target antibody is
detected by simple visual inspection.
B. Detection of Treponema nucleic acids
As noted above, this invention also embraces methods for detecting the
presence of Treponema DNA or RNA in biological samples. These sequences
can be used to detect Treponema in biological samples from hooved animals
such as cattle. A variety of methods of specific DNA and RNA measurement
using nucleic acid hybridization techniques are known to those of skill in
the art (see Sambrook et al., supra).
One method for determining the presence or absence of Treponema DNA in a
sample involves a Southern transfer. Briefly, the digested DNA is run on
agarose slab gels in buffer and transferred to membranes. In a similar
manner, a northern transfer may be used for the detection of Treponema
mRNA in samples of RNA. Hybridization is carried out using labelled
oligonucleotide probes which specifically hybridize to Treponema nucleic
acids. Labels used for this purpose are generally as described for
immunoassays. Visualization of the hybridized portions allows the
qualitative determination of the presence or absence of Treponema genes.
A variety of other nucleic acid hybridization formats are known to those
skilled in the art. For example, common formats include sandwich assays
and competition or displacement assays. Hybridization techniques are
generally described in Nucleic Acid Hybridization, A Practical Approach
(Hades et al., eds. 1985); Gall & Pardue, Proc. Natl. Acad. Sci. U.S.A.,
63:378-383 (1969); and Burnsteil & Jones Nature, 223:582-587 (1969).
Sandwich assays are commercially useful hybridization assays for detecting
or isolating nucleic acid sequences. Such assays utilize a "capture"
nucleic acid covalently immobilized to a solid support and labelled
"signal" nucleic acid in solution. The biological sample will provide the
target nucleic acid. The "capture" nucleic acid and "signal" nucleic acid
probe hybridize with the target nucleic acid to form a "sandwich"
hybridization complex. To be effective, the signal nucleic acid cannot
hybridize with the capture nucleic acid.
The sensitivity of the hybridization assays may be enhanced through use of
a nucleic acid amplification system which multiplies the target nucleic
acid being detected. Examples of such systems include the polymerase chain
reaction (PCR) system and the ligase chain reaction (LCR) system. Other
methods recently described in the art are the nucleic acid sequence based
amplification (NASBA.TM., Cangene, Mississauga, Ontario) and Q Beta
Replicase systems.
An alternative means for detecting Treponema nucleic acids is in situ
hybridization. In situ hybridization assays are well known and are
generally described in Angerer et al., Methods Enzymol., 152:649-660
(1987). In situ hybridization assays use cells or tissue fixed to a solid
support, typically a glass slide. If DNA is to be probed, the cells are
denatured with heat or alkali. The cells are then contacted with a
hybridization solution at a moderate temperature to permit annealing of
labelled Treponema specific probes. The probes are preferably labelled
with radioisotopes or fluorescent reporters.
Exemplary nucleic acid sequences for use in the assays described above
include sequences from the 16S rRNA sequences disclosed here. For
instance, primer and probe sequences derived from the 16S rRNA sequences
of the isolates described herein can be used to amplify and identify
nucleic acids of bovine Treponema in frozen or formalin-fixed foot tissue,
or foot tissue fixed for in situ hybridization. Such 16S rRNA primers are
particularly useful for the diagnosis of PDD.
VII. Pharmaceutical Compositions comprising Treponema
A pharmaceutical composition prepared using anti-Treponema monoclonal
antibodies or fragments thereof as well as Treponema cells, proteins or
their immunogenic equivalents can be used in a variety of pharmaceutical
preparations for the treatment and/or prevention of Treponema infections.
The pharmaceutical compositions are typically used to vaccinate hooved
animals such as cattle, sheep, goats and other animals infected by
Treponema.
The immunogenic whole cell organism, which is employed as the active
component of the present vaccines, consists essentially of inactivated
PDD-associated Treponema spp. These spirochetes can be isolated from
animals affected with PDD, as described above. The spirochetes can be
maintained in infected animals, or in suitable nutrient media. The
immunogenic spirochetes are typically isolated from skin of affected
animals and cultured in defined media.
Another suitable vaccine is a subunit vaccine that elicits antibody and
cell-mediated immunity (CMI) to antigens of bovine Treponema. Experimental
evidence indicates that CMI is an important component of the protective
immune response in cattle. A variety of methods for evaluating the
specificity of the helper and cytotoxic T cell response to selected
antigens in vitro can be used.
To prepare the vaccine, the spirochetes are first separated from the medium
by centrifugation or filtration, or with the use of selective media and
the like. The spirochetes can be treated by a number of methods, including
chemical treatment, to inactivate them. The spirochetes suspensions can be
dried by lyophilization or frozen in an aqueous suspension thereof to
yield inactivated whole cells.
The dried or cultured whole cells are then adjusted to an appropriate
concentration, optionally combined with a suitable vaccine adjuvant, and
packaged for use. Suitable adjuvants include but are not limited to:
surfactants, e.g., hexadecylamine, octadecylamine, lysolectithin,
dimethyl-dioctadecylammonium bromide,
N,N-dioctadecyl-N'-N-bis(2-hydroxyethyl-propane diamine),
methoxyhexadecylglycerol, and pluronic polyols; polyanions, e.g., pyran,
dextran sulfate, dipeptide, dimethylglycine, tuftsin; oil emulsions; and
alum. Finally, the immunogenic product can be incorporated into liposomes
for use in a vaccine formulation, or may be conjugated to polysaccharides
or other polymers.
The absolute weight of the deactivated whole cells varies widely, and
depends upon factors such as age, weight and physical condition of the
subject considered for vaccination. Such factors can be readily determined
by the clinician or veterinarian employing animal models or other test
systems which are all known to the art. A unit dose of the vaccine is
preferably administered parenterally, e.g., by subcutaneous or by
intramuscular injection.
For parenteral administration, the antigen may be combined with a suitable
carrier. For example, it may be administered in water, saline or buffered
vehicles with or without various adjuvants or immunomodulating agents such
as aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate
(alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions,
oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid,
Bordetella pertussis, and the like. Such adjuvants are available
commercially from various sources, for example, Merck Adjuvant 6 (Merck
and Company, Inc., Rahway, N.J.). Other suitable adjuvants are MPL+TDM
Emulsion (RIBBI Immunochem Research Inc. U.S.A.). Other immuno-stimulants
include interleukin 1, interleukin 2 and interferon-gamma. These proteins
can be provided with the vaccine or their corresponding genetic sequence
provided as a functional operon with a recombinant vaccine system such as
vaccinia virus. The proportion of antigen and adjuvant can be varied over
a broad range so long as both are present in effective amounts.
In addition to the Treponema antigen, the vaccine can also include antigens
to other ungulate diseases. For example, the vaccine can include antigens
to ungulate Fusobacterium necrophorum, Porphyromonas levii, and
Dichelobacter nodosus (the organisms that cause interdigital
necrobacillosis, commonly known as foot rot), leptospiral bacteria, bovine
respiratory syncytial virus, bovine Herpes virus, bovine diarhhea virus,
bovine parainfluenza virus, vesicular stomatitis virus, malignant
catarrhal fever virus, blue tongue virus, pseudorabies virus, rabies
virus, rinderpest virus, and Clostridia spp. antigen.
Vaccine compositions of the invention are administered to a cattle, sheep,
horses, or goats susceptible to or otherwise at risk of infection to
induce an immune response against the antigen and thus enhance the
patient's own immune response capabilities. Such an amount is defined to
be an "immunogenically effective amount." In this use, the precise amounts
depend on the judgement of the vaccine manufacturer and prescribing
veterinarian and would include consideration of the patient's state of
health and weight, the mode of administration, the nature of the
formulation, and the like. Generally, on a per-dose basis, the
concentration of the Treponema antigen, typically the whole cell, can
range from about 10 to about 10.sup.9 cells per ungulate patient, or 1
.mu.g to about 100 mg antigen per ungulate patient. For administration to
cattle, a preferable range is from about 10.sup.3 to 10.sup.6 cells or 100
.mu.g to 1 mg antigen per unit dose. A suitable dose volume range is 0.5
to 2.0 ml, preferably about 2 ml. Accordingly, a typical dose for
subcutaneous injection, for example, would comprise 2 ml containing
10.sup.4 cells or 500 .mu.g of antigen.
A variety of vaccination regimens may be effective in immunizing cattle and
other animals. For example, ungulate young and adults can both be
vaccinated, preferably calves. A second immunization will be given 2-4
weeks after initial immunization. Animals that have been previously
exposed to Treponema may require booster injections. The booster injection
is preferably timed to coincide with times of maximal challenge and/or
risk of abortion. Different immunization regimes may be adopted depending
on the judgement of the veterinarian.
Vaccines of the invention may comprise a crude extract of Treponema.
Chemically fixed cells can also be used. As noted above, preferred
vaccines comprise partially or completely purified Treponema protein
preparations. The antigen produced by recombinant DNA technology is
preferred because it is more economical than the other sources and is more
readily purified in large quantities.
In addition to use in recombinant expression systems, the isolated
Treponema gene sequences can also be used to transform viruses that
transfect host cells in animals. Live attenuated viruses, such as vaccinia
or adenovirus, are convenient alternatives for vaccines because they are
inexpensive to produce and are easily transported and administered.
Suitable viruses for use in the present invention include, but are not
limited to, pox viruses, such as canarypox and cowpox viruses, and
vaccinia viruses, alpha viruses, adenoviruses, and other animal viruses.
The recombinant viruses can be produced by methods well known in the art,
for example, using homologous recombination or ligating two plasmids. A
recombinant canarypox or cowpox virus can be made, for example, by
inserting the DNA encoding the Treponema protein or fragments thereof into
plasmids so that they are flanked by viral sequences on both sides. The
DNA encoding Treponema polypeptides are then inserted into the virus
genome through homologous recombination.
Preferentially, a viral vaccine using recombinant vaccinia virus is used. A
vaccine prepared utilizing the gene encoding the Treponema protein
incorporated into vaccinia virus would comprise stocks of recombinant
virus where the gene encoding the Treponema protein is integrated into the
genome of the virus in a form suitable for expression of the gene.
EXAMPLES
The following examples are provided by way of illustration only and not by
way of limitation. Those of skill in the art will readily recognize a
variety of noncritical parameters that could be changed or modified to
yield essentially similar results.
Example I
Isolation of Treponema Strains 1-9185MED and 2-1498
Two strains of Treponema isolates were obtained from PDD infected cattle.
Biopsies or scrapings from PDD-infected cattle foot tissue lesions were
the original source of the cultures. Spirochetes were recovered using
selective broth enrichment with rifampin and/or enrofloxacin (see Walker
et al., Vet. Micro. 47:343-355 (1995)). The cultures were selectively
enriched in OTI broth under anaerobic conditions. To obtain pure cultures
of Treponema, agar blocks with individual spriochete colonies were removed
and placed in OTI broth without antibiotics, propagated for 48 hours,
replated to OTI agar, and assessed for purity.
Morphologic characteristics were assessed on Treponema spirochetes grown on
OTI and 5% sheep blood agar. Morphologic features were evaluated by
darkfield microscopy and transmission electron microscopy.
Enzymatic activity was evaluated using a commercially available system (API
ZYM, Bio Merieux, France). Chromosomal DNA was prepared for restriction
enzyme digestion, digested with EcoRI, HindIII, and HhaI. SDS-PAGE
electrophoresis was used for immunoblot analysis. Whole cell antigens for
production of antisera were obtained using standard procedures from
isolates 2-1498 and 1-9185MED. Antisera was prepared by inoculating
rabbits (Walker et al., Am J. Vet. Res. 49:208-212 (1988)).
Collectively, the morphologic features, antigenic characteristics and the
enzymatic activity of the isolates excluded them from all the known genera
of spirochetes except for Treponema.
Example II
Clinical and Gross Pathological Description of PDD
This example describes the presentation and symptoms of PDD.
Materials and methods
A. Herd management
All cows were Holsteins and were housed outdoors all year in drylot soil
corrals in the Chino and San Jacinto dairy preserves. Average annual
rainfall was 38 cm (range 25 to 89 cm), almost all occurring from January
to March. Accumulated fecal waste was scraped from the corrals during
summer months and from concrete feedbunk platforms and alleyways
periodically throughout the year. Routine footbathing was employed on 1
dairy; copper sulfate was used. The size of the herds ranged from 500 to
2200 cows. Approximate age composition of the milking herds was: 50% at
2-3 years; 40% at 4-5 years; and 10% at 6 years and older. Diet consisted
of a basic ration of alfalfa hay supplemented with various food
commodities and byproducts, such as, cotton seed meal, cotton seed,
soybean meal, rolled grains (corn, barley, wheat), almond hulls and
vitamin-mineral mixtures.
B. Interviews
Interviews were held with 5 commercial hoof trimmers, 8 veterinarians and 4
dairymen in southern California. These individuals gave historical and
general information about approximately 130 dairy herds managed similarly
to those investigated. One veterinarian and 1 hoof trimmer had local
information relative to the past 17 and 25 years, respectively.
Information about PDD was obtained on geographic prevalence, morbidity,
age distribution, anatomic location and gross appearance of lesions and
response of lesions to treatment.
C. Physical examination of lower limbs
Ninety-three cows in 10 herds were selected for examination because of
lameness or grossly visible erosive or papillomatous digital skin lesions.
Age was determined by herd record or ear tag information or tooth eruption
pattern in a total of 49 cows. The majority of cows (n=82 in 9 herds) were
restrained in a tilt chute to facilitate close inspection of all lower
limbs. The remaining 11 cows in 1 herd were only visually examined from
the rear at a distance of .about.1 m as the cows stood in the milking
parlor. A total of 350 feet were examined. Feet were washed with water and
lesions were photographed and recorded. Visual assessments were also made
of corral foot environment (n=7 dairies), trauma of plantar/palmar skin of
the feet (n=68 cows) and the size and shape of the interdigital space (IS)
of hind and fore feet (n=29 cows). Selected lesions (n=85 in 54 cows) were
anaesthetized by locally infiltrating 2% lidocaine into the subcutis and
biopsied for laboratory evaluation by either complete excision or by use
of a 6 mm diameter punch biopsy instrument (Miltex Instrument Co., Lake
Success, N.Y.). Results of histopathologic and bacteriologic evaluations
on biopsy materials are published elsewhere. Read et al., Proc. Int. Sym.
Dis. Rum. Digit 8:156-157 (1994); Read et al., Vet. Rec. 130:59-60 (1992);
Walker et al., Vet. Micro. 47:343-355 (1995).
D. Classification of lesions
Erosive digital skin lesions (n=183 in 93 cows) were classified by anatomic
location and gross appearance; and representative lesions (n.about.85)
were examined histopathologically. Pathologic criteria classified these
lesions into 3 categories: papillomatous digital dermatitis (PDD),
interdigital dermatitis (IDD) and pastern flexural skin fold ulcer (PFSFU)
(Read et al., Proc. Int. Sym. Dis. Rum. Digit 8:156-157 (1994)). Lesions
were histopathologically classified as PDD if they consisted of a
circumscribed plaque of eroded acanthotic epidermis attended by
parakeratotic papillomatous proliferation profusely colonized by
spirochete-dominant bacterial flora, loss of stratum granulosum, invasion
of stratum spinosum by spirochetes and infiltration of neutrophils, plasma
cells, lymphocytes and eosinophils in dermis. Lesions were classified as
IDD if they were located within the IS and had similar histologic
character to PDD, except for demarcated margins and papillomatous change.
Lesions were not classified as IDD if they were grossly confluent with
lesions of PDD. Lesions were classified as PFSFU if they were deep ulcers
attended by pyoderma, folliculitis, furunculosis, serum crusting and
absence of spirochetes. A possible association between PDD and IDD was
examined in 38 feet involved by PDD bordering the interdigital space (IS).
Additional classification was confined to PDD because it was the most
prevalent and painful lesion. These additional studies consisted of
anatomic distribution of lesions (n=129 in 68 cows) and size, shape,
contour, color and surface appearance of lesions (n=134 in 82 cows).
E. Effect of treatments
The effect of various treatments was assessed on a total of 72 lesions of
PDD in 35 cows in 3 herds (herds 4 and 5-7). Treatments consisted of
procaine penicillin G (G. C. Hanford Manufacturing Co., Syracuse, N.Y.),
18,000 units/kg IM BID for 3 days (7 cows); ceftiofur sodium (Naxcel.RTM.,
The Upjohn Co., Kalamazoo, Mich.), 2 mg/kg daily for 3 days (14 cows);
oxytetracycline (Terramycin-343.RTM.Pfizer Animal Health, N.Y.), single
topical application of approximately 5 g of soluble powder bandaged
directly onto a clean lesion for 7 days (3 cows); formadelhyde (Fisher
Scientific, Pittsburg, Pa.), 39% v/v, single topical application (5 cows);
hydrochloric acid (Fisher Scientific, Pittsburg, Pa.), 36% v/v, single
topical application (4 cows); surgical excision (4 cows); and
chlorodifluoromethane, dimethylether (Brand Spray, Stockman Products Ltd.,
Castledown, Isle of Man, UK), 2 minutes of topical cryogenic spray until
lesion and margin were frozen solid and white (1 cow). Two cows which
received no treatment served as untreated controls. Therapeutic response
was assessed at post treatment days: 7 (4 cows), 7 and 14 (23 cows) and 7,
14 and 21 (7 cows). Therapeutic response was considered complete if there
was entire transformation of moist, red, raw, prone-to-bleed, painful
surfaces to dry, dark brown, firm, rubbery, keratinacious, non-painful
surfaces adherent to underlying pink healthy-appearing skin. The
prevalence of recurrent and new lesions was evaluated in 27 of the 35 cows
in herds 6 and 7 that had previously responded to treatment 7-12 weeks
prior to follow-up examination. Another 6 lesions of PDD in 4 cows in herd
4 were reexamined 5 weeks after total surgical excision.
Results
A. Classification of erosive digital skin lesions
Prevalence of PDD, IDD and PFSFU is shown in Table 1. The great majority
(91%) of cows had PDD, whereas smaller numbers had IDD and occasional cows
had PFSFU. Some overlap occurred: 19% and 4% of cows with PDD also had
separate lesions of IDD and PFSFU, respectively. Also, a great majority
(82%) of feet involved by PDD lesions bordering the IS had confluent IDD
lesions extending several millimeters into the IS.
B. Clinical signs of PDD
Cows lame with PDD usually exhibited signs of plantar or palmar pain.
Severely affected cows were reluctant to move; their affected limb was
often held shaking in partial flexion as if in intense pain. Less severely
affected limbs rested or bore weight on the toes, and if unresolved,
hooves became clubbed with atrophy of the bulbs of the heels. Secondary
effects of the lameness included loss of body weight. Little or no diffuse
digital swelling was observed. Fissuring and necrosis of the skin of the
IS were not seen. Heel horn erosion was commonly seen in feet with or
without PDD.
C. Anatomic location of PDD lesions
The anatomic distribution of PDD lesions with respect to affected limb and
anteroposterior, mediolateral and digital locations are shown in Tables 1
to 3. Lesions were confined to the digits and were not observed above the
level of the dewclaws. Lesions exclusively involved the hind limbs in 56
of 68 (82%) of cows (Table 2). Of these, 24 had right limb involvement, 22
had left limb involvement and 10 had both hind limbs involved. The fore
limbs were exclusively involved in 13% of cows and both fore and hind
limbs in 5% of cows. Plantar (or palmar) aspects alone were involved in
84% of cows. The dorsal aspect alone was involved in 13% of cows and
combined plantar/palmar and dorsal aspects were involved in 3% of cows. No
obvious predilection of lesions for medial or lateral digits was observed.
Both medial and lateral digits of an affected individual limb were
involved in 51% of cows: some (31%) of these lesions opposed each other
across the IS, whereas others (19%) confluently involved the entire
commissural skin fold bordering the IS. Either medial or lateral digits of
an individual limb were involved in 10 and 28% of cows, respectively. With
respect to digital site, 76 of 85 (89%) of cows with PDD had lesions
involving skin bordering the IS (Table 1); lesions rarely involved the
abaxial aspects of the digits. Lesions uncommonly involved skin bordering
the base of the bulb of the heel (7 of 85 cows), within the IS (3 of 85
cows), or in plantar pastern flexural skin folds (5 of 85 cows). The
lesions affecting the IS were situated on the crest of a corn (2 cows) or
involved the entire space from plantar to dorsal aspects (1 cow). Six cows
had lesions involving more than 1 site; collectively all 4 sites in table
1 were involved and no pattern was observed. Occasionally, as many as 7
lesions involved an individual limb.
The visual assessments of corral foot environment, trauma of plantar/palmar
skin, and size and shape of the IS gave the following results: hind feet
were submerged deeper in slurry than fore feet during feeding (due to
feedbunk flatform slope), but not at other times; no evidence of
consistent trauma was seen in hind or fore feet; and, the dorsal
two-thirds of the IS of hind and fore feet was markedly more expansive
than the plantar/palmar one-third which was slit-like and difficult to
open manually. No obvious differences were noted in the size and shape of
the plantar region of the IS compared to its palmer counterpart.
D. Gross appearance of PDD
The size, shape, contour, color and surface characteristics of 134 lesions
are presented in Table 3. The majority of lesions were medium to large,
namely, 2-6 cm across at their greatest dimension (88%), circular to oval
(90%), raised (64%), and variable in color and in degree of papillary
proliferation. Washed surfaces were typically either extensively red and
granular (31%) or a composite of white-yellow, grey, brown and/or black
papillary areas interspersed with red granular areas (42%). Lesions
extensively covered by large numbers of papillae comprised 27% of the
total. Papillae were usually filiform; their caliber was about 0.5-1 mm
and their length varied from 1 mm to 3 cm. Small lesions (1 cm across)
were uncommonly observed (12%) and they had similar features to medium and
large lesions except that most had extensively red granular surfaces. A
small proportion of large lesions were "U" shaped because they involved
the entire commissural fold of skin that borders the plantar/palmar IS.
Regardless of size, shape and contour, lesions were characteristically
circumscribed or delineated by a discrete line of raised hyperkeratotic
skin often bearing erect hairs 2-3 times longer than normal. They were
also partially to completely alopecic and their surfaces were moist, prone
to bleed and intensely painful to touch. Lesions proximal and adjacent to
the heel bulb characteristically expanded to involve and replace perioplic
horn. Some lesions undermined the horn of the heel bulb for a distance of
several millimeters but suppurative underrunning of horn was not observed.
Defects in the wall were observed in 2 cows and, in both, the defects were
related to proximal PDD lesions involving the coronary band.
The clinical and gross pathologic features of PDD described here are
essentially identical to those reported for digital dermatitis (DD) in
Canada, Europe, England and Ireland (Basset et al., Vet. Rec. 126:164-165
(1990); Blowey et al., Vet. Rec. 135, 115-117 (1994); Blowey et al., Vet.
Rec. 122:505-508 (1988); Borgmann et al., Can. Vet. 37:35-37 (1983);
Brizzi, Proc. Annu. Conf. Am. Assoc. Bov. Pract. 26:33-37 (1993); Cheli et
al., Proc. Int. Meet. Dis. Cattle 8:208-213 (1974); Gourreau et al., Le
Point Vet 24:49-57 (1992); Zemljic, Proc. Int. Sym. Rum. Digit 8:164-167
(1994)), interdigital papillomatosis in New York (Rebhun, et al., J. Am.
Vet. Med. Assoc. 177:437-440 (1980)) and verrucose dermatitis and digital
papillomatosis in Japan (Kimura et al., J. Vet. Med. Jpn. 46:899-906
(1993)). These features also serve to differentiate PDD from other
specific inflammatory diseases of the digital skin of cattle. The most
striking distinguishing feature noted in the present study was the
anatomic predilection of lesions for hind limbs and skin-horn junctions,
especially those bordering the plantar aspect of the IS. Lesions only
rarely involved the IS per se. Such selective vulnerability appears to be
highly distinctive because it is not reported as a hallmark of other
bovine digital skin diseases (Blowey, In Practice, 85-90 (1992); Greenough
et al., Lameness in Cattle pp. 151-169 (Weaver ed., 2nd ed. 1981); Weaver,
Agri-Practice 9:34-38 (1988)). In addition, other characteristic features
reported here include the propensity of PDD lesions to develop filiform
papillae and that the lesions were intensely painful to touch, prone to
bleed and demarcated by a raised line of hyperkeratotic skin, often
bearing hypertrophied hairs. Overall, these features indicate that PDD,
like DD (Blowey et al., Vet. Rec. 122:505-508 (1988); Greenough et al.,
Lameness in Cattle pp. 151-169 (Weaver ed., 2nd ed. 1981)) represents a
single specific disease entity.
Interdigital necrobacillosis (footrot) differs from PDD because it
primarily involves interdigital skin and is characterized by fissuring,
caseous necrosis of subcutis and diffuse digital swelling (Edmondson,
Proc. Int. Meet Dis. Cattle 8:208-213 (1990); Greenough et al., Lameness
in Cattle pp. 151-169 (Weaver ed., 2nd ed. 1981)). Interdigital dermatitis
(IDD) also primarily involves interdigital skin and causes only mild
lameness (Greenough et al., Lameness in Cattle pp. 151-169 (Weaver ed.,
2nd ed. 1981); Weaver, Agri-Practice 9:34-38 (1988)). Although IDD has
been widely considered a separate entity (Weaver, Agri-Practice 9:34-38
(1988)), its identity has been recently questioned because,
histopathologically, it shares some features in common with DD (Blowey,
Proc. Int. Sym. Dis. Rum. Digit 8:142-154 (1994)); and PDD (Read et al.,
Proc. Int. Sym. Dis. Rum. Digit 8:156-157 (1994)) and it has been
associated with DD in the field (Blowey, Proc. Int. Sym. Dis. Rum. Digit
8:142-154 (1994); Toussaint et al., Vet. Med. Review 2:223-247 (1971)). In
this study it was found that 82% of feet that had PDD bordering the IS
also had contiguous IDD. These observations, as well as the recent
isolation of an identical spirochete in PDD and IDD lesions (Walker et
al., Vet. Micro. 47:343-355 (1995)), indicate that further study is
required to clarify the interrelationships of these 2 entities.
A few cows in this study had involvement of flexural skin folds of the
pastern by either deep ulcers or PDD. The 2 conditions appeared
histologically different (Read et al., Proc. Int. Sym. Dis. Rum. Digit
8:156-157 (1994)) but their predilection for the same site suggests some
commonality in their pathogenesis. Similar ulcers or fissures are reported
to occur on the lower limbs of cattle involved by diffuse exudative
dermatitis housed under prolonged wet unhygienic conditions (McLennan et
al., Aust. Vet. J. 68:76-77 (1991)).
E. Effect of treatments
PDD lesions were highly responsive to parenteral or topical antibiotics or
topical caustic chemicals (Table 4). Sixty-five of 72 lesions involving 30
of 35 cows treated with antibiotics or caustics showed a complete
therapeutic response by post-treatment (PT) day 21. This was characterized
by complete transformation of moist, red, raw, painful prone-to-bleed
surfaces to dry, dark brown, non-painful, tough rubbery keratinacious
surfaces. The keratinacious layer was tightly adherent to underlying
white-pink healthy-appearing skin. These changes were observed in 66% of
cows by PT day 7. By PT day 21, diminution in size and partial restoration
of hair growth were also observed.
Mean therapeutic response times as determined by desiccation and
keratinization of surfaces and absence of pain were slightly longer for
cows treated parenterally with ceftiofur (11.1.+-.4.5 days, n=13) compared
to those treated parenterally with penicillin (8.2.+-.2.5 days, n=7) or
topical applications of oxytetracycline (7 days, n=3), formaldehyde (7
days, n=5) or hydrochloric acid (7 days, n=2). No consistent differences
in response time were observed in lesions of different sizes or at
different sites.
Incomplete responses occurred in 2 cows treated with ceftiofur and in 2
other cows treated with hydrochloric acid. In 1 of the 2 ceftiofur-treated
cows, 2 large raised papillary lesions on one limb were refractory over a
PT period of 73 days despite a second course of antibiotics (parenteral
penicillin); whereas 6 similar medium-sized papillary lesions on the
ipsilateral limb were completely responsive to the first treatment by PT
day 7. In the 2 hydrochloric acid-treated cows, the incomplete responses
were characterized by persistence of small painful deep ulcers. The lesion
treated with cryogenic spray did not respond.
The prevalence of recurrent and new lesions in 27 treated cows in 3 herds
is shown in Table 5. Lesions recurred in 9 cows and new lesions developed
in 4 other cows that had previously responded completely to treatment,
7-12 weeks prior to follow-up examination. Two cows that had recurrent
lesions also had new lesions. The combined rate of recurrence and
new-lesion development in treated cows was 48%. Recurrence and
new-lesion-development were observed in cows treated with either ceftiofur
or penicillin. New lesions occurred in cows treated with hydrochloric acid
and recurrent lesions occurred in cows treated by surgical excision.
The epidemiologic observations indicate that PDD behaves as an infectious
disease, a view also held with respect to DD (Brizzi, Proc. Annu. Conf.
Am. Assoc. Bov. Pract. 26:33-37 (1993); Gourreau et al., Le Point Vet
24:49-57 (1992); Nutter et al., Vet. Rec. 126:200-201 (1990)). The
geographic spread, evidence of contagion, high prevalence in young cows
and high within-herd morbidity observed here are consistent with this
view. In addition, the marked sensitivity of PDD lesions to parenteral or
topical (Britt et al. J. Am. Vet. Med. Assoc. 209:1134-1136 (1996))
antibiotics as well as the presence of intralesional invasive spirochetes
(Read et al., Proc. Int. Sym. Dis. Rum. Digit 8:156-157 (1994); Read et
al., Vet. Rec. 130:59-60 (1992)), provide convincing evidence that
bacteria may play an important role in the pathogenesis of the disease.
Also, the histologic similarity of PDD to yaws, a papillomatous condition
of the feet and lower legs of people living in the tropics caused by
Treponema pallidum subspecies pertenue (Engelkens et al., Int. J. Dermatol
30:77-83 (1991)), adds further support.
In this study, the incidence of PDD in southern California increased in
late spring to early summer. This was later confirmed by an epidemiologic
survey (Rodriguez-Lainz et al., J. Am. Vet. Med. Assoc., 209:1464-1467
(1996)). A subsequent epidemiologic case study of 57 dairies in southern
California revealed that muddiness of corrals was strongly linked to high
PDD prevalence herds (Rodriguez-Lainz et al., Prev. Vet. Med. 28:117-131
(1996)). In the UK and Europe, poor feet hygiene has also been linked with
the occurrence of DD in winter-housed cattle (Blowey, Proc. Int. Sym. Dis.
Rum. Digit 8:142-154 (1994); Blawey et al., Vet. Rec. 122:505-508 (1988);
Nutter et al., Vet. Rec. 126:200-201 (1990)). Suggested predisposing
factors include prolonged contact of the lower limbs with manure-rich
stale slurry (Blowey, Proc. Int. Sym. Dis. Rum. Digit 8:142-154 (1994)), a
foot environment similar to that which we observed during the rainy season
here. However, our observation was confounded by another observation in
this study, namely, that some outbreaks occurred in late fall,
approximately 5 months after the corrals had largely dried out. Others
investigators agree that predisposing factors for outbreaks of DD are not
always clearly evident (Weaver, Proc. 7th Int. Symp. Dis. Rum. Digit;
Zemljic, Proc. Int. Sym. Rum. Digit 8:164-167 (1994)) and still others
report outbreaks associated with excellent hygiene in housed and pastured
cattle. Gourreau et al., Le Point Vet 24:49-57 (1992). As well as
muddiness of corrals, another risk factor revealed by the California
case-control study was the introduction of heifer replacements
(Rodriguez-Lainz et al., Prev. Vet. Med. 28:117-131 (1996)). Other
investigators in the U.S. and Europe also attribute the spread of PDD/DD
to sharing of cows among herds (Gourreau et al., Le Point Vet 24:49-57
(1992)) or introduction of sound heifers from affected herds (Brizzi,
Proc. Annu. Conf. Am. Assoc. Bov. Pract. 26:33-37 (1993); Nutter et al.,
Vet. Rec. 126:200-201 (1990); Weaver, Proc. 7th Int. Symp. Dis. Rum.
Digit; Whittier, Dairy 12-13 (1988)).
The reason why PDD or DD lesions have a high predilection for
plantar/palmar skin bordering the IS is not known. This visual assessment
of corral underfoot environment showed that hind limbs were exposed to
deeper slurry during feeding than were forelimbs, but the significance of
this was not assessed because the preferential site for PDD is almost at
ground level. With respect to the possibility of trauma, no evidence was
found of continual abrasion of either plantar or palmar digital skin.
Visual evaluation of the size and shape of the IS clearly showed that
plantar and palmar regions were slit-like because the bulbs were in close
opposition. The plantar/palmar regions of the IS were, therefore, much
more prone to being continually moist compared to their more open dorsal
counterparts. Since IDD, PDD/DD are favored by a continually moist foot
environment (Blowey, Proc. Int. Sym. Dis. Rum. Digit 8:142-154 (1994);
Greenough et al., Lameness in Cattle, pp. 151-169 (Weaver ed., 2nd ed.
1981); Rodriguez-Lainz et al., J. Am. Vet. Med. Assoc., 209:1464-1467
(1996); Weaver, Agri-Practice 9:34-38 (1988)), this observation may help
to explain why lesions of these entities occur more frequently at these
locations than elsewhere. In this connection, it is significant that a
study on the natural evolution of DD found that 90% of early erosive
lesions began at the plantar border of the IS and then, by proximal local
extension, developed into typical strawberry-like lesions (Morterello et
al., Proc. Int. Sym. Dis. Rum. Digit 8:177-179 (1994)). The plantar/palmar
region of the IS may therefore possess special conducive factors for the
development of PDD, one of which may be a moisture retention property.
In the present study, PDD was observed most frequently in lactating
heifers, a finding also reported by other investigators in the U.S.
(Weaver, Proc. 7th Int. Symp. Dis. Rum. Digit) and Europe (Blawey et al.,
Vet. Rec. 122:505-508 (1988); Brizzi, Proc. Annu. Conf. Am. Assoc. Bov.
Pract. 26:33-37 (1993); Gourreau et al., Le Point Vet 24:49-57 (1992)).
The reason for this apparent age prevalence is unknown but raises the
possibility that infected cows become immune as they age. Against this,
however, is observation of recurrence or new-lesion-development in 48% of
cows reexamined 7-12 weeks after a complete therapeutic response was
observed.
TABLE 1
______________________________________
Classification and anatomic location of 183 erosive digital
skin lesions in 93 Holstein cows in 10 California dairy herds.
No. of No. of
Lesion/Anatomic Site cows.paragraph.
lesions.sctn.
______________________________________
A. Papillomatous digital dermatitis:
1) Proximal border of interdigital space
76 123
2) Proximal border of heel bulb
7 11
3) Interdigital space 3 3
4) Plantar pastern 5 10
B. Interdigital dermatitis
18 27
C. Pastern flexural skin fold ulcer
9 9
______________________________________
.paragraph. = 25 of the 76 cows in category A1 also had lesions listed in
other categories: A2 (2 cows), A2 and A4 (2 cows), A3 (2 cows, B (16 cows
and C (3 cows).
.sctn. = A great majority of A1 lesions extended a few millimeters
distally to involve the skin of the interdigital space.
TABLE 2
______________________________________
Anatomic location of 129 lesions of papillomatous digital
dermatitis in 68 Holstein cows in 8 California dairy herds.*
Cows Lesions
Anatomic location n % n %
______________________________________
Limb:
Right hind 24 35 41 31
Left hind 22 32 38 29
Right and left hind
10 15 27 21
Hind and Forelimbs
3 5 11 9
Forelimbs 9 13 13 10
Plantar/palmar/dorsal:
Plantar/palmar 57 84 115 88
Dorsal 9 13 13 10
Plantar/palmar and dorsal
2 3 2 2
Medial/lateral digits/axial:.paragraph.
Medial 7 10 9 6
Lateral 19 28 31 24
Biaxial 21 31 50 39
Axial 8 12 15 12
Extensive (axial and medial
13 19 25 19
and/or lateral)
______________________________________
* = Herds 2, 4 to 10. In herds 1 and 3, all 4 feet were not systematicall
examined.
.paragraph. = Pertaining to an individual limb.
TABLE 3
______________________________________
Gross appearance of 134 lesions of papillomatous digital
dermatitis in 82 Holstein cows in 9 California dairy herds.*
Number of lesions
Small Medium Large
(1 cm).paragraph.
(2 cm) (3-6 cm)
Lesion characteristic
n (%) n (%) n (%)
______________________________________
Shape:
Circular/oval
16 (12) 52 (39) 53 (39)
U-shaped 0 0 12 (9)
Linear 0 0 1 (1)
Contour:
Concave 4 (3) 0 0
Flat 5 (4) 22 (16) 17 (13)
Raised 7 (5) 30 (22) 49 (37)
Color and Surface:
Red, granular
8 (6) 16 (12) 17 (13)
Red, granular with
5 (4) 21 (16) 29 (22)
yellow/grey papillary
areas
Grey/brown/black,
3 (2) 15 (11) 20 (14)
extensively papillary
______________________________________
* = Herds 1, 2, 4 to 10. In herd 3, lesions were not systematically
examined.
.paragraph. = Greatest dimension across lesion, rounded up/down to neares
cm.
TABLE 4
______________________________________
Effect of various treatments on 72 lesions of papillomatous
digital dermatitis in 35 Holstein cows in 3 California dairy herds.*
No. of lesions
No. of cows assessed
assessed#
Treatment Treated Responded Treated
Responded
______________________________________
Antibiotics
Penicillin G procaine IM
7 7 9 9
Ceftiofur IM 15 13 44 41
Oxytetracycline, topical
3 3 4 4
Topical Caustics
Formaldehyde (39%)
5 5 8 8
Hydrochloric acid (35%)
4 2 6 3
Physical agent
Cryogenic spray
1 0 1 0
______________________________________
* = Herds 5 to 7
# = Therapeutic response was assessed at posttreatment days: 7 (5 cows);
and 14 (23 cows) ; and 7, 14 and 21 (7 cows.) Fiftyfive lesions bordered
the interdigital space; 9 bordered the base of the heel bulb and 8
involved flexural plantar pastern skin fold.
TABLE 5
______________________________________
Prevalence of recurrent and new lesions of papillomatous digital
dermatitis in 27 Holstein cows in 3 herds* that previously completely
responded to treatment 7-12 weeks prior to follow-up examination.
Responsive.paragraph.
Recurrent# New.sctn.
Cows Lesions Cows Lesions
Cows Lesions
Treatment n n n n n n
______________________________________
Antibiotics
Pencillin G procain
3 3 1.sup..vertline.
1 2 4
IM
Cetifur IM 11 30 4.sup..vertline.
6 2 2
Topical Caustics
Formaldehyde (39%)
6 8 0 0 0 0
Hydrochloride acid
3 4 0 0 2 3
(36%)
Surgical excision
4 6 4 6 0 0
Totals 27 51 9 13 6 9
% 33 26 22 18
______________________________________
* = Herds 4, 6 and 7
.paragraph. = Criteria for complete therapeutic response were: absence of
evincible pain and transformation of exudative surfaces to dry brown/blac
rubbery hyperkeratotic layers adherent to whitepink healthyappearing skin
# = Recurrence of a lesion at a previously responsive affected site.
.sctn. = Occurrence of a lesion at a previously nonaffected site
.sup..vertline. = 1 cow also had a new lesion
Example III
Transmission of PDD
Transmission of PDD can be used to demonstrate that vaccinated cattle can
be challenged with PDD. Thus, this is a useful technique to demonstrate
the efficacy of PDD vaccines.
Materials and Methods
A. Animals
Eight 4- to 5-month-old Holstein calves were obtained from a multi-source
calf raising facility in Chino, Calif. They were moved to the San
Bernardino branch laboratory of the California Veterinary Diagnostic
Laboratory System where they were housed on concrete floors in
environmentally controlled isolation rooms for 49 to 111 days. Seven were
female and 1 was a castrated male. Each calf was fed approximately 2 kg of
alfalfa hay and 1.5 kg of mixed grain per day and had ad libitum access to
water. Floors were scraped and hosed clean daily.
B. Experimental design
Two experiments were performed. In experiment I, both hind feet of 6
principals (calves 1-6) and the left hind foot of 2 control (calves 7 & 8)
were constantly maintained in a moist and relatively anaerobic environment
from 6 to 10 days pre-inoculation to the end of the experimental periods.
The foot environment was achieved by wrapping the lower limb, from the
sole to the upper third of the metatarsus, with a polyethylene sheet,
orthopedic cotton and elastic bandage, in that order, followed by placing
the foot inside an impervious plastic boot. The bandages were sprayed with
water until saturated 3 times each day. In experiment II, calves 7 and 8
were utilized as principals, after an acclimation period of 3 weeks
following the end of experiment I. No attempt to constantly maintain moist
and anaerobic foot conditions was performed. The right hind foot of each
calf was inoculated and lightly bandaged keep the inoculum in place. No
boots were applied.
C. Inoculations
Skin of one hind foot of each principal (left hind in calves 1-6 in
experiment I and right hind in calves 7 and 8 in experiment II) was
inoculated at 2 anatomic sites: distal skin-horn junction of the dewclaw
and proximoaxial skin-horn junction of both heels adjacent to the plantar
interdigital space. In 2 principals (calves 1 and 2), skin at the lateral
heel site was mechanically scarified by use of sterile sand paper
immediately before inoculation: Inoculum consisted of chilled homogenate
of PDD lesions which were excised from clinically affected cows 3 to 4
hours prior to use. Activity and identity of the clinical lesions were
confirmed by dark field examination and histopathology. Inoculation was
performed by placing approximately 0.5 g of the homogenate on each skin
site. Inoculum was held in place by a small (1.times.1 cm) piece of
sterile cotton gauze moistened with sterile saline covered by the
prescribed wraps. Inoculation was repeated 7-10 days after the first
inoculation, namely, on PID 7 to 10.
D. Observations
Calves were observed daily for signs of well-being, foot swelling and
lameness. Hind feet were examined weekly for gross lesions. Lesions were
recorded and selected sites were superficially scraped for darkfield
microscopy (calves 1-6) or punch biopsied for bacteriological culture
(Walker et al., Vet. Microbiol. 47:343-355 (1995)) (calves 1 and 2) and
histopathology (calves 1-6). Selected control skin sites were sampled for
darkfield microscopy (calves 1, 3-6) and histopathology (calves 7 and 8 in
experiment I and II). Calves were sampled for blood serum on PID 1 and at
2 weekly intervals thereafter. Serum was stored at -20.degree. C. until
assayed by ELISA for levels of antibody to spirochetes associated with
naturally occurring PDD. Experiments were terminated when lesions
spontaneously resolved, either completely (calves 1-4) or partially
(calves 5 and 6).
Results
A. Experiment I
All principals developed lesions of PDD and all lesions developed at
inoculation sites. Nine of 10 dew claw sites and 2 of 6 heel-interdigital
sites developed lesions. In two principals (calves 1 & 2), non-inoculated
medial dew claw skin developed lesions 7 days after lesions were observed
in their lateral inoculated counterparts. The number of lesions observed
at an inoculation site varied from 1 (calves 3 and 4), 2 to 3 (calves 2, 5
& 6) and 5 (calf 1). The size of lesions varied from 2 to 3 millimeters to
3 centimeters across at their greatest dimension. Small lesions generally
involved dew claw sites and large lesions at heel-interdigital sites. Most
dew claw lesions became grossly apparent at PID 14 to 16 (7 of 11). The
remainder of the dew claw lesions and the heel-interdigital lesions were
first observed at PID 21. Lesions generally increased in size during the
first 2 to 5 weeks after their appearance. Thereafter, they usually
remained static or gradually decreased in size. In one principal, 5
lesions manifested at the heel-interdigital site at various times over a
5-week-period, sometimes resolving and at other times recrudescing. Six
dew claw lesions and the heel-interdigital lesions spontaneously resolved.
Resolution time varied from 33 to 94 days. Five other dew claw lesions
(calves 4-6) were not observed to resolve because the experiments were
terminated at PID 35-54 while the lesions were still active. Resolution
was characterized by centripetal shrinkage, desiccation, keratosis and
loss of pain.
The gross pathologic character of the lesions was uniform irrespective of
site. Early development (PID 14-21) was characterized by matting of the
hairs with dark brown viscid exudate, easily plucked hairs and a diffusely
red moist painful skin surface. By PID 28-35, alopecia was complete, the
eroded surface was red and finely granular and a raised epidermal collar
delineated the lesion from surrounding normal skin. No lesions developed
papilliform proliferations. Local extension of lesions to involve
structures other than skin occurred in 1 principal (calf 2). In this calf,
large lesions encroached upon and replaced perioplic heel horn and, by PID
35, atrophy of heel and clubbing of the hoof was evident.
The histopathologic character of the lesions was also uniform irrespective
of site. Variation was observed in the biopsies according to severity and
chronicity. Lesions in biopsies taken PID 16 to 21 were characterized by:
diffuse loss of stratum corneum; dense colonization of parakeratotic
epidermis and invasion of superficial stratum spinosum and eroded dermal
papillae by long slender spiral bacteria; congestion, thrombosis,
suppuration and necrosis of superficial papillary dermis; acanthosis; and
lymphoplasmacytic perivascular dermatitis. In 7 biopsies taken PID 42 to
71, lesions were similar but more proliferative in character with focal
areas of intense bacterial colonization and inflammation interspersed with
areas of epidermal parakeratosis and hyperkeratosis. Dark field microscopy
findings and bacteriologic culture results of lesions were characteristic
of PDD.
No gross or histopathologic lesions were observed in the control calves
(calves 7 and 8).
B. Experiment II
No gross or histologic lesions were observed in the calves of this
experiment (calves 7 and 8).
The transmission of PDD achieved in this study corroborates for the first
time previously reported anecdotal field observations that the disease is
contagious.
The highly repeatable transmission achieved under controlled environmental
foot conditions, as well as the failure to transmit outside those
conditions, infers that PDD is a multifactorial disease with environmental
as well as infectious causative factors. These experiments identified 2
environmental factors, namely, constant moisture and lack of access to
air. These factors are consistent with findings in a recent epidemiologic
study of PDD in California dairies, namely, that deep muddy corrals
constitute a high risk for contracting the disease.
Example IV
Immunoperoxidase Staining Protocol for PDD in Formalin-fixed,
Paraffin-embedded Tissue
The following protocol was used to determine whether ungulate tissue is
infected with Treponema by detecting Treponema antigen with a specific
antibody. The tissue to be tested was embedded in paraffin and then
sectioned for binding with a specific antibody in situ. The specific
antibody was detected with a labeled secondary antibody. The labeling
pattern was then visualized using a microscope. This protocol was used as
a diagnostic protocol for PDD.
A. Equipment
Micropipette, variable delivery
Microprobe Staining Station
Scale
Water Bath, 37.degree. C.
B. Materials
AEC Substrate, Single Solution (Zymed catalog #00-1111)
Bluing Reagent (Richard Allan catalog #7301)
Coverslips, 24.times.20 (Fisher catalog #12-548-5J)
Crystal Mount, Biomeda (Fisher catalog #BM-M03)
Hematoxylin Solution, Mayer's (Sigma catalog #MHS-16)
Hydrochloric Acid, 1 N (Fisher catalog #SA48-1)
Hydrogen Peroxide, 30% (Sigma catalog #H-1009)
Methanol, Absolute (Fisher catalog #A433-4)
Microscope Slides, Probe-On-Plus (Fisher catalog #15-188-51)
Mounting Medium, Accu-Mount 60 (Baxter catalog #M7630-1)
Pepsin (Sigma catalog #P7000)
Phosphate Buffered Saline (Sigma catalog #1000-3)
Positive Control Tissue
Primary Antibodies, Specific (hyperimmune rabbit sera to Treponema strains
1-9185MED and 2-1498; Walker et al., Vet. Microbiol. 47: 343-355 (1995))
and Non-Specific (normal rabbit serum, Vector Elite)
Rabbit IgG Elite Detection Kit (Vector catalog PK6101)
Reagent Alcohol, ABsolute (Fisher catalog #A962-4)
Tween 20 (Sigma catalog #P-1379)
Xylene (Fisher catalog #X3.sup.P)
C. Reagent preparation
Pepsin solution: 0.6 g pepsin was added to 150 ml 0.01 N hydrochloric acid
(1.5 ml 1 N HCl+150 ml deionized water). The solution was placed in a
37.degree. C. water bath for 30 minutes before use.
Hydrogen peroxide solution, 3%: 15 ml 30% hydrogen peroxide was added to
135 ml absolute methanol.
Phosphate buffered saline (PBS): one package PBS was added to one liter
deionized water. Check pH and, if necessary, adjust to 7.4.
PBS/Tween: 600 .mu.l (12 drops) Tween was added to 240 ml BPS. The
10.times. Automation Buffer (100 ml) in deionized water (900 ml) may
substitute for PBS/Tween.
PBS/Tween/Alcohol: 100 ml reagent grade absolute alcohol was added to 900
ml PBS/Tween.
Normal goat serum, Vector Elite: Three drops stock normal goat serum was
added to ten ml PBS/Tween.
Primary antibodies: Diluted in PBS to 1:400.
Biotinylated secondary antibody, Vector Elite: Three drops stock normal
goat serum were added to ten ml PBS/Tween. Mixed, one drop biotinylated
antibody added.
ABC reagent, Vector Elite: Two drops of reagent "A" were added to five ml
PBS/Tween. Mix and add two drops reagent "B", mix immediately. ABC reagent
was allowed to stand 30 minutes before use.
AEC Single Solution: Used directly from bottle. If solution is colored,
discard.
D. Procedure
For each specific primary antibody, two slides from each paraffin block to
be tested and two positive control slides are needed. Tissue was sectioned
at three to four microns, and mounted on far right-hand side of "Probe On
Plus" microscope slides. One slide was labeled with the specific primary
antibody name and dilution (i.e., anti-Treponema antibody 1-9185 1:400)
and the other with the nonspecific primary antibody name and dilution
(i.e., NRS 1:400). Both slides were labeled with the case accession and
block number (or control lot number), detection system (i.e., ERK), and
run date. The sections were allowed to dry overnight. Unless otherwise
noted, all incubations and reagents are used at room temperature. The
buffers and detection kit were stored in a refrigerator. The buffers are
checked for gross contamination before use. Primary antibodies are stored
at -7.degree. C.
The slides were placed in a conventional slide holder, and deparaffinized
for five minutes in each of three changes of xylene. The slides are placed
in two changes of reagent grade absolute alcohol, allowing three minutes
for each change. The slide holder was agitated for the first ten seconds
of each of these and subsequent reagent changes, excluding those changes
where the Microprobe handle was used.
The slides were immersed in 3% hydrogen peroxide solution for ten minutes.
The sections were rehydrated on the slides for two minutes in each change
of 95, 80% and 70% reagent grade alcohols, and two changes of deionized
water.
The slides were placed in pepsin solution for 15 minutes at 37.degree. C.
The slides were rinsed in four changes of deionized water, one minute each
change.
The slides were placed face to face (section to section) in the Microprobe
handle. A blank Probe-On-Plus slide was used to pair-up any unpaired
slides. If multiple stains were being performed, record which slide in the
Microprobe handle receives which primary antibody.
Rinse/blot cycles were performed using PBS/Tween and paper towels.
Rinse/blot cycles followed using PBS/Tween/Alcohol. At the end of these
rinse/blot cycles, all air bubbles should have been removed from between
the slides.
Normal goat serum was applied to the slides for 20 minutes. All reagents
should be applied to the edge of each set of slides to ensure that the all
parts of the tissue receive reagent. To compensate for evaporation, slides
were allowed to stand in a pool of the reagent, or placed in a moist
chamber.
Normal goat serum was blotted from the slides and the specific and
non-specific primary antibodies applied for one hour. Ten blot/rinse
cycles were performed with PBS/Tween/Alcohol.
Biotinylated secondary antibody was applied to the slides for 15 minutes.
Blot/rinse cycles were performed with PBS/Tween/Alcohol.
ABC reagent was applied for 15 minutes. Ten blot/rinse cycles were
performed with PBS/Tween.
AEC reagent was applied for three minutes. Three blot/rinse cycles were
performed with deionized water, slides were transferred to conventional
slide holder, and deionized water was applied for three minutes.
The slides were counterstained with filtered Mayer's hematoxylin for 90
seconds and then rinsed for two minutes in running tap water.
The slides were blued for one minute in hematoxylin bluing reagent and
rinsed in two changes of deionized water, two minutes each.
Crystal/Mount medium was applied over sections and the slides were placed
on a 70-80.degree. C. hot plate for a minimum of ten minutes. The slides
were removed from hot plate and allow to cool. The coverslip was mounted
with Accu-Mount mounting medium.
The control and test slides were evaluated for staining. Only the specific
primary antibody slide should show red staining of the antigen being
stained.
Example V
Vaccine Preparation and Immunization
A. Origin and Cultivation of PDD-Associated Treponema spp.
PDD-associated Treponema spp. have been recognized as likely etiologic
agents involved in the pathogenesis of PDD. These spirochetes were first
isolated and cultivated in oral treponeme isolation (OTI) broth at
37.degree. C. (Walker et al., Vet. Microbiol. 47:343-355 (1995)). In
addition, a modified Barbour-Stoenner Kelly (BSK) medium was used to
culture the organisms at 30-37.degree. C. in microaerophilic to anaerobic
conditions to a density between 1.times.10.sup.4 to 1.times.10.sup.9
spirochetes/ml. The modified medium was prepared by the addition of 0.15%
agarose (Seakem LE; FMC Corp., Rockland, Me.) to the BSK medium. The BSK
medium was prepared as disclosed by Barbour, Isolation and cultivation of
Lyme disease spirochetes, in Lyme Disease, First International Symposium,
pp. 71-75 (Steele et al., eds., (1984)).
B. Vaccine preparation and immunization
The vaccine was prepared by suspending organisms to a specific optical
density in 0.5% formalized physiological saline solution. This cellular
preparation was devoid of viable cells. Neither culture or the injection
of the cell preparation used in rabbits provided evidence of viable
spirochetes. Non-pregnant, female, New Zealand White rabbits were injected
with a single dose of vaccine subcutaneously followed by 5 intravenous
doses of vaccine at 3 day intervals. Rabbits were bled prior to
immunization and one week after the last injection. No adjuvant was used.
For vaccination of male Holstein calves, spirochete antigen was prepared
by growing spirochetes in OTI broth for 48 hours at 37.degree. C. Cultures
were washed in physiological saline solution and suspended to a
concentration equivalent to an OD.sub.550 of 0.4 in 0.5% formalized
saline. The suspension was stored at -70.degree. C. until used. Calves
were initially given 1 subcutaneous injection followed by 6 intravenous
immunizations at 3 day intervals. Calves were bled prior to immunization
and one week after the last vaccination. No adjuvant was used.
TABLE 6
______________________________________
Active Immunization of Rabbits with PDD-associated Treponenia spp.
strain 1-9185-MED and 2-1498 PDD.
1-9185MED 2-1498
______________________________________
Pre-vaccination ELISA
<1:100 1:400
titer to homologous
spirochete
Post-vaccination ELISA
>1:6400 >1:6400
titer to homologous
spirochete
______________________________________
Titers are determined as the lowest dilution at which the optical density
was two times that of the optical density obtained when no serum was used
in the ELISA.
TABLE 7
______________________________________
Active immunization of calves with PDD-associated Treponema spp.
strain 1-9185 MED and 2-1498.
1-9185MED 2-1498
______________________________________
Pre-vaccination ELISA
1:200 1:200
titer to homologous
spirochete
Post-vaccination ELISA
>1:6400 1:6400
titer to homologous
spirochete
______________________________________
Titers are determined as the lowest dilution at which the optical density
was two times that of the optical density obtained when no serum was used
in the ELISA.
The data summarized in Table 7 indicates that a repeated immunization with
PDD-associated Treponema spp results in boosting the levels of serum
antibodies to those spirochetes in the cow. The use of a plurality of
vaccinations is expected to increase the duration of immunity conferred
and it is expected that a vaccine comprising inactivated or attenuated
Treponema spirochetes will be effective to actively immunize susceptible
mammals against PDD.
Furthermore, it is expected that the efficacy of vaccines based on
PDD-associated Treponema spp. will be increased by employing immunogenic
fractions derived therefrom by methods which are known to the art. For
example, the treponemal outer envelope which surrounds the protoplasmic
cylinder of spirochetes can be readily extracted (Klaviter et al., Acta.
Trop. 36:123 (1979)). This fraction may provide immunogens which impart an
equal or greater resistance to PDD infection when employed as the active
component of vaccines prepared in accord with the present invention.
Recombinant outer surface proteins may also be used as an immunogen.
All publications and patent applications cited in this specification are
herein incorporated by reference as if each individual publication or
patent application were specifically and individually indicated to be
incorporated by reference.
Although the foregoing invention has been described in some detail by way
of illustration and example for purposes of clarity of understanding, it
will be readily apparent to one of ordinary skill in the art in light of
the teachings of this invention that certain changes and modifications may
be made thereto without departing from the spirit or scope of the appended
claims.
__________________________________________________________________________
# SEQUENCE LISTING
- <160> NUMBER OF SEQ ID NOS: 20
- <210> SEQ ID NO 1
<211> LENGTH: 1413
<212> TYPE: DNA
<213> ORGANISM: Treponema sp.
<220> FEATURE:
#2-1498THER INFORMATION: 16S rRNA for spirochete
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (43)
<223> OTHER INFORMATION: n = unknown
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (270)
<223> OTHER INFORMATION: n = unknown
- <400> SEQUENCE: 1
- acgctggcgg cgcgtcttaa gcatgcaagt cgaacggcaa ggnaggagct tg - #cttctccc
60
- ctagagtggc ggactggtga gtaacgcgtg ggtgatctgc ccttaagatg gg - #gatagctc
120
- ctagaaatag gaggtaatac cgaatacgct tatacggata aagccgtata ag - #gaaaggag
180
- gctacggcct tgcttgagga tgagcccgcg tcccattatg cttgttggtg ag - #gtaacggc
240
- ttaccaaggc gacgatgggt atccggcctn agagggtgga cggacacatt gg - #gactgaga
300
- tacggcccaa actcctacgg gaggcagcag ctaagaatat tccgcaatgg ac - #ggaagtct
360
- gacggagcga cgccgcgtgg acgaagaagg ccgaaaggtt gtaaagttct tt - #tgccgatg
420
- aagaataaga ggatgaggga atgcgtcctt gatgacggta gtcgagcgaa ta - #agccccgg
480
- ctaattacgt gccagcagcc gcggtaacac gtaaggggcg agcgttgttc gg - #aattattg
540
- ggcgtaaagg gcacgcaggc gggttggtaa gcctgatgtg aaatactcaa gc - #ttaacttg
600
- agaattgcat tgggtactgc cagtcttgaa tcacggaggg gaaaccggaa tt - #ccaagtgt
660
- aggggtggaa tctgtagata tttggaagaa caccggtggc gaaggcgggt tt - #ctggccga
720
- tgattgacgc tgaggtgcga aggtgtgggg agcaaacagg attagatacc ct - #ggtagtcc
780
- acacagtaaa cgatgtacac taggtgttgg ggcaagagct tcagtgccgg cg - #caaacgca
840
- ataagtgtac cgcctgggga gtatgcccgc aagggtgaaa ctcaaaggaa tt - #gacggggg
900
- cccgcacaag cggtggagca tgtggtttaa ttcgatgata cgcgaggaat ct - #tacctggg
960
- tttgacatca aaagcaatat tatagagata tggtagcgta gcaatacggc tt - #ttgacagg
1020
- tgctgcatgg ctgtcgtcag ctcgtgccgt gaggtgttgg gttaagtccc gc - #aacgagcg
1080
- caacccctac tgtcagttgc taacaggtaa tgctgaggac tctggcggaa ct - #gccgatga
1140
- caaatcggag gaaggtgggg atgacgtcaa gtcatcatgg cccttatgtc ca - #gggctaca
1200
- cacgtgctac aatggttgct acaaagtgaa gcgagactgt gaggttaagc aa - #atcgcaaa
1260
- aaagcaatcg tagttcggat tgaagtctga aactcgactt catgaagttg ga - #atcgctag
1320
- taatcgcaca tcagcacggt gcggtgaata cgttcccggg ccttgtacac ac - #cgcccgtc
1380
# 1413 gggt acccgaagtc gcc
- <210> SEQ ID NO 2
<211> LENGTH: 1417
<212> TYPE: DNA
<213> ORGANISM: Treponema sp.
<220> FEATURE:
#1-9185MEDR INFORMATION: 16S rRNA for spirochete
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (627)
<223> OTHER INFORMATION: n = unknown
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (963)
<223> OTHER INFORMATION: n = unknown
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (1007)
<223> OTHER INFORMATION: n = unknown
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (1408)
<223> OTHER INFORMATION: n = unknown
- <400> SEQUENCE: 2
- gagcttgctc ttaccctaga gtggcggact ggtgagtaac gcgtaggtga cc - #tgccctga
60
- agatggggat agctagtaga aatattagat aataccgaat gtgcttatac gg - #ataaagcc
120
- gtataaggaa aggagctacg gctccgcttt aggatgggcc tgcgtcccat ta - #gcttgttg
180
- gtgaggtaac ggcccaccaa ggcgacgatg ggtatccggc ctgagagggt ga - #acggacac
240
- attgggactg agatacggcc caaactccta cgggaggcag cagctaagaa tc - #ttccgcaa
300
- tggacgaaag tctgacggag cgacgccgcg tgaatgaaga aggctgaaaa gt - #tgtaaaat
360
- tcttttgcag atgaagaata aggagatgag ggaatgcatc ttcgatgacg gt - #aatcatgc
420
- gaataagggg cggctaatta cgtgccagca gccgcggtaa cacgtaagcc cc - #aagcgttg
480
- ttcggaatta ttgggcgtaa agggcatgta ggcggttatg taagcctgat gt - #gaaatcta
540
- cgagcttaac tcgtaaactg cattgggtac tgcgtaactt gaatcacgga gg - #ggaaaccg
600
- gaattccaag tgtaggggtg gaatctngta gatatttgga agaacaccgg tg - #gcgaaggc
660
- gggtttctgg ccgatgattg acgctgagat gcgaaggtgc ggggagcaaa ca - #ggattaga
720
- taccctggta gtccgcacag taaacaatgt acactaggcg ttggagcaag aa - #cttcagtg
780
- ccgacgcaaa cgcattaagt gtaccgcctg ggaaagtatg cccgcaaggg tg - #aaactcaa
840
- aggaattgac gggggcccac acaagcggtg gagcatgtgg tttaattcga tg - #atacgcga
900
- ggaaccttac ctgggtttga catcaagagt aatggtatag agatatatca gc - #gtarcaat
960
- acngactctt gacaggtgct gcatggctgt cgtcagctcg tgccgtnaag gt - #gttgggtt
1020
- aagtcccgca mcragcgcaa cccctactgc cagttactaa cacgtaaagg tt - #gaggactc
1080
- tggcggaact gccgatgaca aatcggagga aggtggggat gacgtcaagt ca - #tcatggcc
1140
- cttacgtcca gggctacaca cgtgctacaa tggttgctac aaatcgaagc ga - #cgccscga
1200
- ggccaagcaa aacgcaaaaa agcaatcgta gtccggattg aagtctgaaa ct - #cgacttca
1260
- tgaagttgga atcgctagta atcgcacats arywcstgyg cggtgaatac st - #tcctgggc
1320
- cttgtacaca ccgcccgtca caccatycga gtcgagggta cccgaagccg yt - #agtcwgac
1380
# 1417 tgtm crmgagyncg cytggwa
- <210> SEQ ID NO 3
<211> LENGTH: 1462
<212> TYPE: DNA
<213> ORGANISM: Treponema sp.
<220> FEATURE:
#7-2009THER INFORMATION: 16S rRNA for spirochete
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (274)
<223> OTHER INFORMATION: n = unknown
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (1115)
<223> OTHER INFORMATION: n = unknown
- <400> SEQUENCE: 3
- acgaacgctg gcggcgcgtc ttaagcatgc aagtcgaacg gcaagggagg ag - #cttgcttc
60
- tcccctagag tggcggactg gtgagtaacg cgtgggtgat ctgcccttaa ga - #tggggata
120
- gctcctagaa ataggaggta ataccgaata cgcttatacg gataaagccg ta - #taaggaaa
180
- ggaggctacg gccttgcttg aggatgagcc cgcgtcccat tatgcttgtt gg - #tgaggtaa
240
- cggcttacca aggcgacgat gggtatccgg cctnagaggg tggacggaca ca - #ttgggact
300
- gagatacggc ccaaactcct acgggaggca gcagctaaga atattccgca at - #ggacggaa
360
- gtctgacgga gcgacgccgc gtggacgaag aaggccgaaa ggttgtaaag tt - #cttttgcc
420
- gatgaagaat aagaggatga gggaatgcgt ccttgatgac ggtagtcgag cg - #aataagcc
480
- ccggctaatt acgtgccagc agccgcggta acacgtaagg ggcgagcgtt gt - #tcggaatt
540
- attgggcgta aagggcacgc aggcgggttg gtaagcctga tgtgaaatac tc - #aagcttaa
600
- cttgagaatt gcattgggta ctgccagtct tgaatcacgg aggggaaacc gg - #aattccaa
660
- gtgtaggggt ggaatctgta gatatttgga agaacaccgg tggcgaaggc gg - #gtttctgg
720
- ccgatgattg acgctgaggt gcgaaggtgt ggggagcaaa caggattaga ta - #ccctggta
780
- gtccacacag taaacgatgt acactaggtg ttggggcaag agcttcagtg cc - #ggcgcaaa
840
- cgcaataagt gtaccgcctg gggagtatgc ccgcaagggt gaaactcaaa gg - #aattgacg
900
- ggggcccgca caagcggtgg agcatgtggt ttaattcgat gatacgcgag ga - #atcttacc
960
- tgggtttgac atcaaaagca atattataga gatatggtag cgtagcaata cg - #gcttttga
1020
- caggtgctgc atggctgtcg tcagctcgtg ccgtgaggtg ttgggttaag tc - #ccgcaacg
1080
- agcgcaaccc ctactgtcag ttgctaacag gtaangctga ggactctggc gg - #aactgccg
1140
- atgacaaatc ggaggaaggt ggggatgacg tcaagtcatc atggccctta tg - #tccagggc
1200
- tacacacgtg ctacaatggt tgctacaaag tgaagcgaga ctgtgaggtt aa - #gcaaatcg
1260
- caaaaaagca atcgtagttc ggattgaagt ctgaaactcg acttcatgaa gt - #tggaatcg
1320
- ctagtaatcg cacatcagca cggtgcggtg aatacgttcc cgggccttgt ac - #acaccgcc
1380
- cgtcacacca tccgagttga gggtacccga agtcgccagt ctaacccgta ag - #ggagggcg
1440
# 1462ggc aa
- <210> SEQ ID NO 4
<211> LENGTH: 1366
<212> TYPE: DNA
<213> ORGANISM: Treponema sp.
<220> FEATURE:
#9-3301THER INFORMATION: 16S rRNA for spirochete
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (78)
<223> OTHER INFORMATION: n = unknown
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (334)
<223> OTHER INFORMATION: n = unknown
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (1006)
<223> OTHER INFORMATION: n = unknown
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (1121)
<223> OTHER INFORMATION: n = unknown
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (1200)
<223> OTHER INFORMATION: n = unknown
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (1228)
<223> OTHER INFORMATION: n = unknown
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (1250)
<223> OTHER INFORMATION: n = unknown
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (1262)
<223> OTHER INFORMATION: n = unknown
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (1271)
<223> OTHER INFORMATION: n = unknown
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (1300)
<223> OTHER INFORMATION: n = unknown
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (1313)
<223> OTHER INFORMATION: n = unknown
- <400> SEQUENCE: 4
- agcttgcttc tcccctagag tggcggactg gtgagtaacg cgtgggtgat ct - #gcccttaa
60
- gatggggata gctcctanaa ataggaggta ataccgaata cgcttatacg ga - #taaagccg
120
- tataaggaaa ggaggctacg gccttgcttg aggatgagcc cgcgtcccat ta - #tgcttgtt
180
- ggtgaggtaa cggcttacca aggcgacgat gggtatccgg cctgagaggg tg - #gacggaca
240
- cattgggact gagatacggc ccaaactcct acgggaggca gcagctaaga at - #attccgca
300
- atggacggaa gtctgacgga gcgacgccgc gtgnacgaag aaggccgaaa gg - #ttgtaaag
360
- ttcttttgcc gatgaagaat aagaggatga gggaatgcgt ccttgatgac gg - #tagtcgag
420
- cgaataagcc ccggctaatt acgtgccagc agccgcggta acacgtaagg gg - #cgagccgt
480
- tgttcggaat tattgggcgt aaagggcacg caggcgggtt ggtaagcctg at - #gtgaaata
540
- ctcaagctta acttgagaat tgcattgggt actgccagtc ttgaatcacg ga - #ggggaaac
600
- cggaattcca agtgtagggg tggaatctgt agatatttgg aagaacaccg gt - #ggcgaagg
660
- cgggtttctg gccgatgatt gacgctgagg tgcgaaggtg tggggagcaa ac - #aggattag
720
- ataccctggt agtccacaca gtaaacgatg tacactaggt gttggggcaa ga - #gcttcagt
780
- gccggcgcaa acgcaataag tgtaccgcct ggggagtatg cccgcaaggg tg - #aaactcaa
840
- aggaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga tg - #atacgcga
900
- ggaatcttac ctgggtttga catcaaaagc aatattatag agatatggta gc - #gtagcaat
960
- acggcttttg acaggtgctg catggctgtc gtcagctcgt gccgtnaggt gt - #tgggttaa
1020
- gtcccgcaac gagsgcaacc cctactgtca gttgctaaca ggtaacgctg ag - #gactctgg
1080
- cggaactgcc gatgacaaat cggaggaagg tggggatgac ntcaagtcat ca - #tggccctt
1140
- atgtccaggg ctacacacgt gctacaatgg ttgctacaaa gtgaagcgag ac - #tgtgaggn
1200
- taagcaaatc gcaaaaaagc aatcgtantt cggattgaaa tctgaaactn ga - #cttcatga
1260
- anttggaatc nctagtaatc gcacatcagc acggtgcggn gaataccttc cc - #nggccttg
1320
# 1366cca tccgagttga gggtacccga agtcgc
- <210> SEQ ID NO 5
<211> LENGTH: 1405
<212> TYPE: DNA
<213> ORGANISM: Treponema sp.
<220> FEATURE:
#9-3143THER INFORMATION: 16S rRNA for spirochete
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (42)
<223> OTHER INFORMATION: n = unknown
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (414)
<223> OTHER INFORMATION: n = unknown
- <400> SEQUENCE: 5
- ctgcggcgcg tcttaagcat gcaagtcgaa cggcaaggta anagcttgct ct - #taccctag
60
- agtggcggac tggtgagtaa cgcgtaggtg acctgccctg aagatgggga ta - #gctagtag
120
- aaatattaga taataccgaa tgtgcttata cggataaagc cgtataagga aa - #ggagctac
180
- ggctccgctt taggatgggc ctgcgtccca ttagcttgtt ggtgaggtaa cg - #gcccacca
240
- aggcgacgat gggtatccgg cctgagaggg tgaacggaca cattgggact ga - #gatacggc
300
- ccaaactcct acgggaggca gcagctaaga atcttccgca atggacgaaa gt - #ctgacgga
360
- gcgacgccgc gtgaatgaag aaggctgaaa agttgtaaaa ttcttttgca ga - #tnaagaat
420
- aaggagatga gggaatgcat cttcgatgac ggtaatcatg cgaataaggg gc - #ggctaatt
480
- acgtgccagc agccgcggta acacgtaagc cccaagcgtt gttcggaatt at - #tgggcgta
540
- aagggcatgt aggcggttat gtaagcctga tgtgaaatct acgagcttaa ct - #cgtaaact
600
- gcattgggta ctgcgtaact tgaatcacgg aggggaaacc ggaattccaa gt - #gtaggggt
660
- ggaatctgta gatatttgga agaacaccgg tggcgaaggc gggtttctgg cc - #gatgattg
720
- acgctgagat gcgaaggtgc ggggagcaaa caggattaga taccctggta gt - #ccgcacag
780
- taaacaatgt acactaggcg ttggagcaag agcttcagtg ccgacgcaaa cg - #cattaagt
840
- gtaccgcctg ggaagtatgc ccgcaagggt gaaactcaaa ggaattgacg gg - #ggcccaca
900
- caagcggtgg agcatgtggt ttaattcgat gatacgcgag gaaccttacc tg - #ggtttgac
960
- atcaagagta atggtataga gatatatcag cgtagcaata cgactcttga ca - #ggtgctgc
1020
- atggctgtcg tcagctcgtg ccgtgaggtg ttgggttaag tccctgcaac ga - #gcgcaaac
1080
- ccctactgcc agttactaac acgtaaaggt tgaggactct ggcggaactg cc - #gatgacaa
1140
- atcggaggaa ggtggggatg acgtcaagtc atcatggccc ttacgtccag gg - #ctacacac
1200
- gtgctacaat ggttgctaca aatcgaagcg acgccgcgag gccaagcaaa ac - #gcaaaaaa
1260
- gcaatcgtag tccggattga agtctgaaac tcgacttcat gaagttggaa tc - #gctagtaa
1320
- tcgcacatca gcacggtgcg gtgaatacgt tcctgggcct tgtacacacc gc - #ccgtcaca
1380
# 1405 tacc cgaaa
- <210> SEQ ID NO 6
<211> LENGTH: 1414
<212> TYPE: DNA
<213> ORGANISM: Treponema sp.
<220> FEATURE:
#9-3528THER INFORMATION: 16S rRNA for spirochete
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (12)
<223> OTHER INFORMATION: n = unknown
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (18)
<223> OTHER INFORMATION: n = unknown
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (38)
<223> OTHER INFORMATION: n = unknown
- <400> SEQUENCE: 6
- aacgaacgct gncggcgngt cttaagcatg caagtcgnac ggcaaggtaa ga - #gcttgctc
60
- ttaccctaga gtggcggact ggtgagtaac gcgtaggtga cctgccctga ag - #atggggat
120
- agctagtaga aatattagat aataccgaat gtgcttatac ggataaagcc gt - #ataaggaa
180
- aggagctacg gctccgcttt aggatgggcc tgcgtcccat tagcttgttg gt - #gaggtaac
240
- ggcccaccaa ggcgacgatg ggtatccggc ctgagagggt gaacggacac at - #tgggactg
300
- agatacggcc caaactccta cgggaggcag cagctaagaa tcttccgcaa tg - #gacgaaag
360
- tctgacggag cgacgccgcg tgaatgaaga aggctgaaaa gttgtaaaat tc - #ttttgcag
420
- atgaagaata aggagatgag ggaatgcatc ttcgatgacg gtaatcatgc ga - #ataagggg
480
- cggctaatta cgtgccagca gccgcggtaa cacgtaagcc ccaagcgttg tt - #cggaatta
540
- ttgggcgtaa agggcatgta ggcggttatg taagcctgat gtgaaatcta cg - #agcttaac
600
- tcgtaaactg cattgggtac tgcgtaactt gaatcacgga ggggaaaccg ga - #attccaag
660
- tgtaggggtg gaatctgtag atatttggaa gaacaccggt ggcgaaggcg gg - #tttctggc
720
- cgatgattga cgctgagatg cgaaggtgcg gggagcaaac aggattagat ac - #cctggtag
780
- tccgcacagt aaacaatgta cactaggcgt tggagcaaga gcttcagtgc cg - #acgcaaac
840
- gcattaagtg taccgcctgg gaagtatgcc cgcaagggtg aaactcaaag ga - #attgacgg
900
- gggcccacac aagcggtgga gcatgtggtt taattcgatg atacgcgagg aa - #ccttacct
960
- gggtttgaca tcaagagtaa tggtatagag atatatcagc gtagcaatac ga - #ctcttgac
1020
- aggtgctgca tggctgtcgt cagctcgtgc cgtgaggtgt tgggttaagt cc - #cgcaacga
1080
- gcgcaacccc tactgccagt tactaacacg taaaggttga ggactctggc gg - #aactgccg
1140
- atgacaaatc ggaggaaggt ggggatgacg tcaagtcatc atggccctta cg - #tccagggc
1200
- tacacacgtg ctacaatggt tgctacaaat cgaascgacg ccgcgaggcc aa - #gcaaaacg
1260
- caaaaaagca atcgtagtcc ggattgaagt ctgaaactcg acttcatgaa gt - #tggaatcg
1320
- ctagtaatcg cacatcagca cggtgcggtg aatacgttcc tgggccttgt ac - #acaccgcc
1380
# 1414 cgag ggtacccgaa gccg
- <210> SEQ ID NO 7
<211> LENGTH: 1400
<212> TYPE: DNA
<213> ORGANISM: Treponema sp.
<220> FEATURE:
#9-3379THER INFORMATION: 16S rRNA foe spirochete
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (21)
<223> OTHER INFORMATION: n = unknown
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (24)
<223> OTHER INFORMATION: n = unknown
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (26)
<223> OTHER INFORMATION: n = unknown
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (122)
<223> OTHER INFORMATION: n = unknown
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (173)..(174)
<223> OTHER INFORMATION: n = unknown
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (182)
<223> OTHER INFORMATION: n = unknown
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (606)
<223> OTHER INFORMATION: n = unknown
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (980)
<223> OTHER INFORMATION: n = unknown
<220> FEATURE:
<221> NAME/KEY: modified.sub.-- base
<222> LOCATION: (1293)
<223> OTHER INFORMATION: n = unknown
- <400> SEQUENCE: 7
- taagcatgca agtcgaacgg naananagga gcttgcttct ctcctagagt gg - #cggactgg
60
- tgaggaacac gtgggtaatc tacccttaag atggggatag ctgctagaaa ta - #gcaggtaa
120
- tnccgaatac actcagtgct tcataagggg tattgaggaa aggaagctac gg - #nnttcgct
180
- tnaggatgag cttgcgtccc attagctagt tggtgaggta aaggcccacc aa - #ggcgacga
240
- tgggtatccg gcctgagagg gtgatcggac acattgggac ttgagatacg gc - #ccaaactc
300
- ctacgggagg cagcagctaa gaatattccg caatggacgg aagtctgacg ga - #gcgacgcc
360
- gcgtggatga agaaggctga aaagttgtaa aatccttttg ttgatgaaga at - #aagggtga
420
- gagggaatgc tcatctgatg acggtaatcg acgaataagc cccggctaat ta - #cgtgccag
480
- cagccgcggt aacacgtaag gggcgagcgt tgttcggaat tattgggcgt aa - #agggcatg
540
- taggcggtta tgtaagcctg atgtgaaatc ctggggctta accctagaat ag - #cattgggt
600
- actgtntaac ttgaattacg gaagggaaac tggaattcca agtgtagggg tg - #gaatctgt
660
- agatatttgg aagaacaccg gtggcgaagg cgggtttctg gccgataatt ga - #cgctgaga
720
- tgcgaaagtg tggggatcga acaggattag ataccctggt agtccacacc gt - #aaacgatg
780
- tacactaggt gttggggcaa gagcttcagt gccaaagcaa acgcgataag tg - #taccgcct
840
- ggggagtatg cccgcaaggg tgaaactcaa aggaattgac gggggcccgc ac - #aagcggtg
900
- gagcatgtgg tttaattcga tggtacgcga ggaaccttac ctgggtttga ca - #tctagtag
960
- aaggtcttag agataaggcn gggtagcaat accctgctag acaggtgctg ca - #tggctgtc
1020
- gtcagctcgt gccgtgaggt gttgggttaa gtcccgcaac gagcgcaacc cc - #tactgcca
1080
- gttactaaca ggtaaagctt gaggactctg gcggaactgc cgatgacaaa tc - #ggaggaag
1140
- gtggggatga cgtcaagtca tcatggccct tatgtccagg gctacacacg tg - #ctacaatg
1200
- gttgctacaa agcgaagcaa gaccgtaagg tggagcaaac cgcaaaaaag ca - #atcgtagt
1260
- tcggattgaa gtctgaaact cgacttcatg aanttggaat cgctagtaat cg - #cgcatcag
1320
- cacggcgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac ca - #tccgagtt
1380
# 140 - #0
- <210> SEQ ID NO 8
<211> LENGTH: 1446
<212> TYPE: DNA
<213> ORGANISM: Treponema sp.
- <400> SEQUENCE: 8
- acgctggcgg cgcgtcttaa gcatgcaagt cgaacggcaa gagaggagct tg - #cttctctc
60
- ctagagtggc ggactggtga ggaacacgtg ggtaatctac ccttaagatg gg - #gatagctg
120
- ctagaaatag caggtaatac cgaatacact cagtgcttca taaggggtat tg - #aggaaagg
180
- aagctacggc ttcgcttgag gatgagcttg cgtcccatta gctagttggt ga - #ggtaaagg
240
- cccaccaagg cgacgatggg tatccggcct gagagggtga tcggacacat tg - #ggactgag
300
- atacggccca aactcctacg ggaggcagca gctaagaata ttccgcaatg ga - #cggaagtc
360
- tgacggagcg acgccgcgtg gatgaagaag gctgaaaagt tgtaaaatcc tt - #ttgttgat
420
- gaagaataag ggtgagaggg aatgctcatc tgatgacggt aatcgacgaa ta - #agccccgg
480
- ctaattacgt gccagcagcc gcggtaacac gtaaggggcg agcgttgttc gg - #aattattg
540
- ggcgtaaagg gcatgtaggc ggttatgtaa gcctgatgtg aaatcctggg gc - #ttaaccct
600
- agaatagcat tgggtactgt gtaacttgaa ttacggaagg gaaactggaa tt - #ccaagtgt
660
- aggggtggaa tctgtagata tttggaagaa caccggtggc gaaggcgggt tt - #ctggccga
720
- taattgacgc tgagatgcga aagtgtgggg atcgaacagg attagatacc ct - #ggtagtcc
780
- acaccgtaaa cgatgtacac taggtgttgg ggcaagagct tcagtgccaa ag - #caaacgcg
840
- ataagtgtac cgcctgggga gtatgcccgc aagggtgaaa ctcaaaggaa tt - #gacggggg
900
- cccgcacaag cggtggagca tgtggtttaa ttcgatggta cgcgaggaac ct - #tacctggg
960
- tttgacatct agtagaaggt cttagagata aggccgggta gcaataccct gc - #tagacagg
1020
- tgctgcatgc ctgtcgtcag ctcgtgccgt gaggtgttgg gttaagtccc gc - #aacgagcg
1080
- caacccctac tgccagttac taacaggtaa agcttgagga ctctggcgga ac - #tgccgatg
1140
- acaaatcgga ggaaggtggg gatgacgtca agtcatcatg gcccttatgt cc - #agggctac
1200
- acacgtgcta caatggttgc tacaaagcga agcaagaccg taaggtggag ca - #aaccgcaa
1260
- aaaagcaatc gtagttcgga ttgaagtctg aaactcgact tcatgaagtt gg - #aatcgcta
1320
- gtaatcgcgc atcagcacgg cgcggtgaat acgttcccgg gccttgtaca ca - #ccgcccgt
1380
- cacaccatcc gagttggggg tacccgaagt cgcttgtcta acctgcaaag ga - #ggacggtg
1440
# 1446
- <210> SEQ ID NO 9
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
#Sequence:forwardMATION: Description of Artificial
primer (E. coli base 8 to 27)
- <400> SEQUENCE: 9
# 20 tcag
- <210> SEQ ID NO 10
<211> LENGTH: 19
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
#Sequence:reverseMATION: Description of Artificial
#1510 to 1492(E. coli complement bases
- <400> SEQUENCE: 10
# 19 ctt
- <210> SEQ ID NO 11
<211> LENGTH: 17
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
#Sequence:forwardMATION: Description of Artificial
primer
- <400> SEQUENCE: 11
# 17 c
- <210> SEQ ID NO 12
<211> LENGTH: 19
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
#Sequence:reverseMATION: Description of Artificial
primer
- <400> SEQUENCE: 12
# 19 tcc
- <210> SEQ ID NO 13
<211> LENGTH: 19
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
#Sequence:forwardMATION: Description of Artificial
primer
- <400> SEQUENCE: 13
# 19 ggg
- <210> SEQ ID NO 14
<211> LENGTH: 19
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
#Sequence:forwardMATION: Description of Artificial
primer
- <400> SEQUENCE: 14
# 19 ttg
- <210> SEQ ID NO 15
<211> LENGTH: 18
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
#Sequence:forwardMATION: Description of Artificial
primer
- <400> SEQUENCE: 15
# 18 gg
- <210> SEQ ID NO 16
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
#Sequence:forwardMATION: Description of Artificial
primer
- <400> SEQUENCE: 16
# 20 tggg
- <210> SEQ ID NO 17
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
#Sequence:reverseMATION: Description of Artificial
primer
- <400> SEQUENCE: 17
# 20 tact
- <210> SEQ ID NO 18
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
#Sequence:forwardMATION: Description of Artificial
primer
- <400> SEQUENCE: 18
# 20 cgcg
- <210> SEQ ID NO 19
<211> LENGTH: 15
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
#Sequence:reverseMATION: Description of Artificial
primer
- <400> SEQUENCE: 19
# 15
- <210> SEQ ID NO 20
<211> LENGTH: 18
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
#Sequence:reverseMATION: Description of Artificial
primer
- <400> SEQUENCE: 20
# 18 ct
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